Generation of two induced pluripotent stem cell lines (LVPEIi007-B, LVPEIi008-B) from patients harboring homozygous mutation in ABCA4 (c.6088C>T) using non-integrative Sendai virus-based approach.

Mutations in ABCA4 gene leads to the most common form of an inherited retinal disease namely, the Stargardt disease, type 1. Here, we report the generation of two different patient-specific induced pluripotent stem cell lines (LVPEIi007-B and LVPEIi008-B), carrying an identical homozygous mutation, (c.6088C>T) within the exon 44 of ABCA4 gene. These lines were generated by the reprogramming of patient-specific dermal fibroblasts, using the integration-free, Sendai viral vectors. Both lines were stably expanded and expressed the stemness and pluripotency markers, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

Generation of two induced pluripotent stem cell lines (LVPEIi004-A and LVPEIi005-A) from probands with Leber Congenital Amaurosis 2 (LCA2) and harboring mutations in RPE65.

Leber Congenital Amaurosis 2 is an early onset retinal dystrophy that occurs due to mutation in RPE65 gene. Here, we report the generation of two patient specific induced pluripotent stem cell lines harboring nonsense mutations in exon 7 (c.646A > T) and exon 9 (c.992G > A) of RPE65 gene, respectively, which leads to premature translational termination and formation of defective protein. These lines were generated by the reprogramming of human dermal fibroblast cells using integration-free, episomal constructs expressing stemness genes. The stable lines maintained a normal karyotype, expressed the key stemness factors, underwent trilineage differentiation, and maintained their genetic identity and genomic integrity.

Generation of Leber congenital amaurosis, type 12 patient-specific induced pluripotent stem cell line (LVPEIi006-A), harboring a homozygous mutation in RD3.

Leber congenital amaurosis (LCA) is a congenital, early onset, autosomal recessive inherited retinal disease (IRD). This report describes an LCA12 patient-specific iPSC line (LVPEIi006-A), generated by the reprogramming of dermal fibroblasts using integration-free episomal plasmids.This disease-specific iPSC model carries a homozygous point mutation in RD3, within the donor splice site at the end of exon 2 (c.296 + 1G > A). The stable line at passage 15 has displayed a normal colony morphology, expressed multiple stemness and pluripotency markers, lost all transgenes, differentiated into cell types of all three germ layers, and maintained a normal karyotype.

RPE65 mutations in Leber congenital amaurosis, early-onset severe retinal dystrophy, and retinitis pigmentosa from a tertiary eye care center in India.

INTRODUCTION: Mutations in the retinal pigment epithelial 65 kilodalton protein (RPE65) gene are associated with various inherited retinal diseases (IRDs), including Leber congenital amaurosis (LCA), early-onset severe retinal dystrophy (EOSRD), and retinitis pigmentosa (RP). We screened for mutations in RPE65 in a series of Indian patients with these IRDs to determine the frequency/types of mutations and to describe the associated phenotypes. MATERIALS AND METHODS: Diagnosis of LCA, EOSRD, and RP was made by standard and pre-defined criteria. Patients were evaluated by clinical, retinal imaging, and electrophysiological parameters. Genomic DNA from patients and available family members were used for identifying mutations by direct Sanger sequencing of the RPE65 gene or targeted NGS gene panel for IRDs covering 260+ genes. Variations detected were tested in healthy control populations and for co-segregation with the disease in available family members. RESULTS: Mutations were found in eight patients, out of 220 total cases screened, all homozygous for the respective mutant alleles. Seven patients had mutations leading to premature termination codons and one patient had a missense change. The onset of visual loss ranged from birth to <2 years of life. At presentation, RPE mottling in the background retina was present in all cases with macular involvement in five cases with or without vascular attenuation and optic disc pallor. CONCLUSION: RPE65 mutations in this series were found in 3.6% of cases associated with severe, early-onset disease, with consistent RPE mottling and variable manifestations with regard to the extent of disc pallor, arteriolar attenuation, and appearance of the macula.

Updates on congenital hereditary endothelial dystrophy.

Congenital hereditary endothelial dystrophy (CHED) is a rare genetic corneal disorder causing progressive cornea clouding and significant visual impairment. CHED remains a leading indication for pediatric corneal transplantation despite its infrequency, particularly in regions with high consanguinity rates like Southeast Asia. Identifying the Solute Carrier Family 4 Member 11 (SLC4A11) gene as the genetic basis of CHED has led to the discovery of it's various genetic variations. However, a comprehensive understanding of its clinical-genetic correlation, pathophysiology, and optimal management is ongoing. This review aims to consolidate current knowledge about CHED, covering its genetic origins, pathophysiological mechanisms, clinical presentation, and management strategies. Surgical intervention, such as penetrating keratoplasty (PK), Descemet stripping automated endothelial keratoplasty (DSAEK), and Descemet membrane endothelial keratoplasty (DMEK), remains the primary treatment. DSAEK and DMEK offer advantages over PK, including quicker visual recovery, reduced complications, and longer graft survival, especially in the pediatric age group. The timing of surgical interventions depends on disease severity, age at presentation, comorbidities, and visual potential. Elevated oxidative stress in CHED corneal tissue suggests potential benefits from anti-inflammatory drugs to rescue mutated endothelial cells. Considering the limitations of corneal graft surgeries, exploring novel gene-based molecular therapies are essential for future management. Early diagnosis, appropriate surgical interventions, amblyopia control, and genetic counseling for predictive analysis are pivotal for optimizing CHED management. A multidisciplinary approach involving ophthalmologists, researchers, and genetic counselors is essential for precise diagnosis and optimal care for CHED patients.

Identification and in silico analysis of a spectrum of SLC4A11 variations in Indian familial and sporadic cases of congenital hereditary endothelial dystrophy.

BACKGROUND: Congenital hereditary endothelial dystrophy (CHED) is a rare form of corneal dystrophy caused by SLC4A11 gene variations. This study aims to find the genetic alterations in SLC4A11, in two Indian familial CHED cases with affected members n = 3 and n = 2 respectively and five sporadic CHED cases using direct sequencing, followed by in silico analysis and characterization of the identified variants. RESULTS: All three affected members of the first CHED family were identified with a novel homozygous c.1514C > G (p.Ser489Trp) variation while second family showed presence of a compound heterozygous variation c.529A > C (p.Arg161Arg) + c.2461insT (p.Val805fs). Among five sporadic cases, two showed novel changes, homozygous c.1487G > T (p.Ser480Ile) and c.620-2A > G, while the other one had previously reported homozygous c.2653C > T (p.Arg869Cys) variation. The remaining two cases did not reveal the presence of SLC4A11-related pathogenic variations. The identified variations were excluded from the Indian control (n = 80). In silico analysis using homology-based protein modeling and pathogenicity prediction tools, which revealed these alterations as pathogenic, changing their protein stability, local flexibility, residue contact clashes, and the hydrogen bond interactions. CONCLUSIONS: This study contributed to the CHED mutational spectrum, adding four novel variations and confirming a previously reported one. It demonstrates different type of variations in CHED cases, including coding, non-coding, homozygous, synonymous, and compound heterozygous variations. The identified variations revealed different degrees of pathogenic effects in silico. Moreover, two sporadic cases could not be identified with pathogenic variation emphasizing the involvement of other genes or genetic mechanisms.

Update on the genetics of corneal endothelial dystrophies.

Corneal endothelial dystrophies are a heterogeneous group of diseases with different modes of inheritance and genetic basis for each dystrophy. The genes associated with these diseases encode transcription factors, structural components of the stroma and Descemet membrane, cell transport proteins, and others. Congenital hereditary endothelial dystrophy (CHED) is associated with mutations in two genes, OVOL2 and SLC4A11, for dominant and recessive forms of CHED, respectively. Mutations in three genes are known to cause posterior polymorphous corneal dystrophy (PPCD). They are OVOL2 (PPCD1), ZEB1 (PPCD3), and GRHL1 (PPCD4). The PPCD2 locus involving the collagen gene COL8A2 on chromosome 1 is disputed due to insufficient evidence. Mutations in the COL8A2 gene are associated with early-onset Fuchs' endothelial corneal dystrophy (FECD). Several genes have been associated with the more common, late-onset FECD. Alterations in each of these genes occur in a fraction of patients, and the most prevalent genetic alteration in FECD patients across the world is a triplet repeat expansion in the TCF4 gene. Knowledge of the genetics of corneal endothelial dystrophies has considerably advanced within the last decade and has contributed to better diagnosis of these dystrophies as well as opened up the possibility of novel therapeutic approaches based on the molecular mechanisms involved. The functions of genes identified to date provide insights into the pathogenic mechanisms involved in each disorder.

Genetics of Inherited Retinal Diseases in Understudied Populations.

Retinitis pigmentosa is one of the major forms of inherited retinal dystrophy transmitted in all Mendelian and non-Mendelian forms of inheritance. It involves the loss of retinal photoreceptor cells with severe loss of vision or blindness within the first 2 decades of life. RP occurs at a relatively high prevalence in India and is often associated with consanguinity in certain South Asian communities where this practice is customary. This review describes the studies that have been published with regard to genetics of retinitis pigmentosa in India and neighboring South Asian countries. These populations have been understudied in these aspects although to a variable degree from one country to another. Genetic studies on RP in India have been carried out with a range of methods aimed at detecting specific mutations, to screening of candidate genes or selected genomic regions, homozygosity mapping to whole genome sequencing. These efforts have led to a molecular genetic characterization of RP in Indian families. Similar studies on large extended families from Pakistan have provided insight into several novel genes underlying the pathogenesis of these diseases. The extreme degree of clinical and genetic heterogeneity of RP renders it challenging to identify the associated genes in these populations, and to translate the research output towards better management of the disease, as there are no unifying genetic features that are characteristic of any population so far.

Identification of Key Genes and Pathways in Persistent Hyperplastic Primary Vitreous of the Eye Using Bioinformatic Analysis.

Background: The failure of the embryonic hyaloid vascular system to regress naturally causes persistent hyperplastic primary vitreous (PHPV), a congenital eye disease. PHPVs molecular pathway, candidate genes, and drug targets are unknown. The current paper describes a comprehensive analysis using bioinformatics to identify the key genes and molecular pathways associated with PHPV, and to evaluate potential therapeutic agents for disease management. Methods: The genes associated with PHPV were identified using the pubmed2ensembl text mining platform. GeneCodis was employed to evaluate the Gene Ontology (GO) biological process terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Search Tool for the Retrieval of Interacting Genes (STRING) constructed a protein-protein interaction (PPI) network from the text mining genes (TMGs) in Cytoscape. The significant modules were clustered using Molecular Complex Detection (MCODE), and the GO and KEGG analysis for the hub genes were analyzed with the Database of Annotation, Visualization and Integrated Discovery (DAVID) tool. ClueGO, CluePedia, and ShinyGo were used to illustrate the functions and pathways of the clustered hub genes in a significant module. The Drug-Gene Interaction database (DGIdb) was used to evaluate drug-gene interactions of the hub genes to identify potential PHPV drug candidates. Results: A total of 50 genes associated with PHPV were identified. Overall, 35 enriched GO terms and 15 KEGG pathways were discovered by the gene functional enrichment analysis. Two gene modules were obtained from the PPI network constructed with 31 nodes with 42 edges using MCODE. We selected 14 hub genes as core candidate genes: TP53, VEGFA, SMAD2, CDKN2A, FOXC, FZD4, LRP5, KDR, FZD5, PAX6, MYCN, NDP, PITX2, and PAX2, primarily associated with camera-type eye morphogenesis, pancreatic cancer, the apoptotic process involved in morphogenesis, and the VEGF receptor signaling pathway. We discovered that 26 Food and Drug Administration (FDA)-approved drugs could target 7 of the 14 hub genes. Conclusions: In conclusion, the results revealed a total of 14 potential genes, 4 major pathways, 7 drug gene targets, and 26 candidate drugs that could provide the basis of novel targeted therapies for targeted treatment and management of PHPV.

Macular Corneal Dystrophy: An Updated Review.

Macular Corneal Dystrophy is an autosomal recessive form of corneal dystrophy due to a mutation in CHST6 gene, which results in abnormal proteoglycan synthesis. There is accumulation of abnormal glycosaminoglycans in the corneal stroma and endothelium. The deposition results in progressive loss of corneal transparency and visual acuity. The histopathology shows characteristic alcian blue positive deposits. Management in the cases with visual loss requires keratoplasty either full thickness or lamellar. The decision about the ideal type of keratoplasty depends on age and pre-operative clinical features. Although prognosis after keratoplasty is good, recurrences can occur. Future research should be targeted towards gene therapy in this condition.

Review: Intraflagellar transport proteins in the retina.

Intraflagellar transport (IFT) is an essential process in all organisms that serves to move proteins along flagella or cilia in either direction. IFT is performed by IFT particles, which are multiprotein complexes organized into two subcomplexes, A and B. The IFT proteins form interactions with each other, with cargo proteins, and with membranes during the transport process. Several IFT proteins are expressed in many parts of the retina, such as the outer plexiform and outer nuclear layers, and function in the transport of photoreceptor proteins between the inner and outer segments. Mutants of IFT protein genes have been characterized in model organisms such as Chlamydomonas, C. elegans, zebrafish, and the mouse. These mutants have defective ciliogenesis or abnormalities in retinal photoreceptors. Mutations in IFT genes are associated with syndromic and non-syndromic forms of retinal disease in humans, frequently with early onset of disease.

The spermatogenesis-associated protein-7 (SPATA7) gene - an overview.

BACKGROUND: The spermatogenesis-associated protein-7 (SPATA7) gene encodes a ciliary protein that is expressed in the photoreceptors and in spermatocytes. Mutations in the SPATA7 gene are associated with congenital and early-onset forms of retinal dystrophy. METHODS: Papers and review articles on SPATA7 were retrieved from the PubMed database using the search terms "SPATA7" and "spermatogenesis-associated protein 7". Those that were relevant to retinal disease or to the function of the SPATA7 gene were selected for review. RESULTS: The SPATA7 locus was mapped as LCA3 to chromosome 14, and the gene identified by screening of all genes in the refined genomic interval. Mutations in SPATA7 are associated with Leber congenital amaurosis (LCA) and early-onset retinitis pigmentosa. There are no clear-cut correlations between the genotypes and phenotypes in SPATA7-associated disease, and phenotypic heterogeneity occurs among patients with the same mutation. The SPATA7 protein is expressed in the photoreceptor connecting cilia. Murine models of Spata7 knockout have been useful in understanding the role of this gene in the retina at the cellular and molecular levels. CONCLUSION: Most of the mutations in the SPATA7 are nonsense or frameshifts and are predicted to lead to loss of function. Clinical heterogeneity is often seen in patients with SPATA7 mutations. Animal models of SPATA7 knockout indicate that the protein has a key role in organizing the ciliary protein complexes.

Genetic Markers for Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis in the Asian Indian Population: Implications on Prevention.

This review attempts to collate all the studies performed in India or comprising a population originating from India and to find out if there is an association between the HLA (human leucocyte antigen) type of individual and development of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) subsequent to medication use. The authors performed a PubMed search of all articles published in English from 2009 to 2019 for articles that studied HLA type in patients who developed SJS/TEN after intake of a specific drug in the Asian Indian population or in individuals of Asian Indian origin. The selection criteria were satisfied by a total of 11 studies that reported HLA associations with specific drugs, which induced SJS/TEN, mainly anti-epileptic drugs, and cold medicine/non-steroidal anti-inflammatory drugs. These studies involved a small number of patients, and hence, there is limited evidence to conclude if these associations can be extrapolated to a larger population of the same ethnicity. Similar multi-center studies need to be conducted with a larger sample size to confirm these associations. This would have implications in policy making and for understanding the potential of using genetic markers as a screening tool before prescribing a drug to a patient, which might make them susceptible to developing a potentially life-threatening disease such as SJS/TEN. This is possibly the only mode of primary prevention for this potentially fatal severe cutaneous adverse drug reaction.

Coexistence of Congenital Hereditary Endothelial Dystrophy and Fuchs Endothelial Corneal Dystrophy Associated With SLC4A11 Mutations in Affected Families.

PURPOSE: To evaluate parents of probands affected with autosomal recessive congenital hereditary endothelial dystrophy (CHED) for clinical signs of Fuchs endothelial corneal dystrophy (FECD) and to determine the genotypes of the SLC4A11 gene in the probands and their parents. METHODS: This study involved 9 patients affected with CHED from 8 families. The parents of such probands were examined to investigate for possible signs of FECD although they did not present with any visual complaints. Blood samples were collected after obtaining consent, from all 9 cases and the parents of each proband, for genetic analysis. Genomic DNA isolated from blood leukocytes was used for genetic analysis. Screening of the coding regions of the SLC4A11 gene for mutations was carried out by standard procedures using polymerase chain reaction (PCR) amplification and sequencing. Sequences were checked against the human SLC4A11 reference sequence to detect alterations and in normal control samples available in the laboratory. RESULTS: The probands had characteristic signs of CHED, whereas one of the parents of each of the probands showed early signs of FECD, characterized by the presence of guttae in the central cornea corroborative with the diagnosis of FECD, and were otherwise asymptomatic at that time. The CHED-affected probands were homozygous for various SLC4A11 mutations, and their parents were heterozygous for the same. The mutations included missense mutations in 6 probands and nonsense mutations in 2 cases. CONCLUSIONS: Heterozygosity for SLC4A11 mutations in the parents of children with autosomal recessive CHED appears to be a risk factor for the development of FECD in these cases.

Therapeutic avenues for hereditary forms of retinal blindness.

Hereditary retinal diseases, known as retinal degenerations or dystrophies, are a large group of inherited eye disorders resulting in irreversible visual loss and blindness. They develop due to mutations in one or more genes that lead to the death of the retinal photoreceptor cells. Till date, mutations in over 200 genes are known to be associated with all different forms of retinal disorders. The enormous genetic heterogeneity of this group of diseases has posedmany challenges in understanding the mechanisms of disease and in developing suitable therapies. Therapeutic avenues that are being investigated for these disorders include gene therapy to replace the defective gene, treatment with neurotrophic factors to stimulate the growth of photoreceptors, cell replacement therapy, and prosthetic devices that can capture light and transmit electrical signals through retinal neurons to the brain. Several of these are in process of human trials in patients, and have shown safety and efficacy of the treatment. A combination of approaches that involve both gene replacement and cell replacement may be required for optimum benefit.

A Fluorescent Quantitative Multiplex PCR Method to Detect Copy Number Changes in the RB1 Gene.

Copy number changes comprising deletions or insertions involving single or multiple exons of a gene are known to occur in a significant proportion of cases in retinoblastoma. The protocol described here involves a two-step quantitative multiplex PCR process which is suitable for the detection of such mutations in the RB1 as well as in other genes. This is achieved through the use of suitable gene-specific primers designed to amplify individual exons, with universal tags attached to the 5' end of each primer. These tagged primers are used in the first step of PCR of the RB1 gene in patients. The second step is carried out through the use of "universal" primers complementary to the tag sequences alone. This technique facilitates the detection of fluorescent PCR products from multiple exons through the use of a single fluorescent tagged primer.

Association of Human Leukocyte Antigen Class 1 genes with Stevens Johnson Syndrome with severe ocular complications in an Indian population.

Stevens Johnson syndrome (SJS) is part of a spectrum of adverse drug reactions resulting in the destruction of skin, mucous membranes, and the ocular surface. A similar, more severe form of the disorder included in this spectrum is toxic epidermal necrolysis (TEN). Approximately 35% of patients suffer chronic sequelae such as vascularization, corneal scarring, conjunctival inversion to the cornea, keratinization, symblepharon, scarring of the palpebral conjunctiva, trichiasis, and severe dry eye. We focused on 80 Indian patients with SJS/TEN with severe ocular complications (SOC) and investigated the association of alleles at HLA -A, HLA-B and HLA-C loci; the controls were 50 healthy Indian volunteers. Genotyping at HLA-A, HLA-B, and HLA-C loci showed a significant positive association with HLA-A*33:03, HLA-B*44:03, and HLA-C*07:01 alleles, and a significant negative association with HLA-B*57:01 and HLA-C*06:02. This indicates that HLA-A*33:03, HLA-B*44:03 and HLA-C*07:01 are risk alleles, and HLA-B*57:01 and HLA-C*06:02 are protective alleles in this population. We also found that the haplotypes consisting of HLA-B*44:03 and HLA-C*07:01 were strongly associated with SJS/TEN with SOC in our Indian population (p = 1.1 × 10-7, odds ratio = 11.0). Describing the association of the haplotype could facilitate the understanding of increased risk factors for developing SJS/TEN with SOC.