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.

A missense mutation in ASRGL1 is involved in causing autosomal recessive retinal degeneration.

Inherited retinal dystrophies are a group of genetically heterogeneous conditions with broad phenotypic heterogeneity. We analyzed a large five-generation pedigree with early-onset recessive retinal degeneration to identify the causative mutation. Linkage analysis and homozygosity mapping combined with exome sequencing were carried out to map the disease locus and identify the p.G178R mutation in the asparaginase like-1 gene (ASRGL1), segregating with the retinal dystrophy phenotype in the study pedigree. ASRGL1 encodes an enzyme that catalyzes the hydrolysis of L-asparagine and isoaspartyl-peptides. Studies on the ASRGL1 expressed in Escherichia coli and transiently transfected mammalian cells indicated that the p.G178R mutation impairs the autocatalytic processing of this enzyme resulting in the loss of functional ASRGL1 and leaving the inactive precursor protein as a destabilized and aggregation-prone protein. A zebrafish model overexpressing the mutant hASRGL1 developed retinal abnormalities and loss of cone photoreceptors. Our studies suggest that the p.G178R mutation in ASRGL1 leads to photoreceptor degeneration resulting in progressive vision loss.

Human ßA3/A1-crystallin splicing mutation causes cataracts by activating the unfolded protein response and inducing apoptosis in differentiating lens fiber cells.

ßγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the ßA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant ßA3/A1-crystallin protein. Transgenic expression of mutant ßA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del ßA3/A1-crystallin with resultant activation of the stress signaling pathway - unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts.

IKZF1, a new susceptibility gene for cold medicine-related Stevens-Johnson syndrome/toxic epidermal necrolysis with severe mucosal involvement.

BACKGROUND: Stevens-Johnson syndrome (SJS) and its severe form, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucous membranes, including the ocular surface, oral cavity, and genitals. These reactions are very rare but are often associated with inciting drugs, infectious agents, or both. OBJECTIVE: We sought to identify susceptibility loci for cold medicine-related SJS/TEN (CM-SJS/TEN) with severe mucosal involvement (SMI). METHODS: A genome-wide association study was performed in 808 Japanese subjects (117 patients with CM-SJS/TEN with SMI and 691 healthy control subjects), and subsequent replication studies were performed in 204 other Japanese subjects (16 cases and 188 control subjects), 117 Korean subjects (27 cases and 90 control subjects), 76 Indian subjects (20 cases and 56 control subjects), and 174 Brazilian subjects (39 cases and 135 control subjects). RESULTS: In addition to the most significant susceptibility region, HLA-A, we identified IKZF1, which encodes Ikaros, as a novel susceptibility gene (meta-analysis, rs4917014 [G vs. T]; odds ratio, 0.5; P = 8.5 × 10(-11)). Furthermore, quantitative ratios of the IKZF1 alternative splicing isoforms Ik1 and Ik2 were significantly associated with rs4917014 genotypes. CONCLUSION: We identified IKZF1 as a susceptibility gene for CM-SJS/TEN with SMI not only in Japanese subjects but also in Korean and Indian subjects and showed that the Ik2/Ik1 ratio might be influenced by IKZF1 single nucleotide polymorphisms, which were significantly associated with susceptibility to CM-SJS/TEN with SMI.

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.

Generation and characterization of a Stargardt's disease-specific induced pluripotent stem cell line (LVPEIi008-A) with a homozygous nonsense mutation in exon 44 of ABCA4.

The Stargardt's Disease, Type 1 (STGD1) is associated with the loss of function mutations in ABCA4. This gene codes for a retina-specific, ATP-binding cassette (ABC) family transporter, involved in the transport of the key visual cycle intermediate, all-trans-retinaldehyde (atRAL), across the photoreceptor cell membranes. Here, we report the establishment of a patient-specific, iPSC line (LVPEIi008-A), that carries a homozygous nonsense mutation at (c.6088C > T) position, within exon 44 of ABCA4. The patient-specific skin fibroblasts were reprogrammed using episomal plasmids and the stably expanding iPSC line expressed the key stemness and pluripotency markers, maintained its chromosomal integrity and tested negative for mycoplasma.

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.

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.

Mutational screening of Indian families with hereditary congenital cataract.

PURPOSE: To screen for pathogenic mutations in ten candidate genes in Indian families diagnosed with autosomal recessive and autosomal dominant cataracts. METHODS: Families with two or more affected individuals with bilateral familial congenital/developmental cataract were ophthalmically evaluated, and blood samples were obtained. Genomic DNA extracted from the blood leukocytes was screened with PCR amplification of the exons and the flanking intronic regions of various genes selected for analysis. The amplified products were subjected to single strand conformation polymorphism (SSCP) analysis. The variants in SSCP analysis were subjected to bidirectional sequencing by automated methods. RESULTS: We identified four novel sequence changes that cosegregated with the disease phenotype in each family and were absent in at least 50 ethnically matched unrelated normal controls. These changes include a homozygous missense change of c.649G>A (Val196Met) in GJA8/connexin 50 (Cx50) in a family with autosomal recessive cataract, two heterozygous missense changes, c.658C>T (Pro199Ser) in GJA8/Cx50 and c.589C>T (Pro197Ser) in GJA3/connexin 46 (Cx46) in two separate families with autosomal dominant cataract, and a silent change ( c.84G>A/p.Val28Val, predicted to result in the creation of a new potential branch point) in GJA8 one family with an autosomal dominant inheritance of cataract. Of the four novel mutations identified, three mutations, Val196Met (GJA8), Pro199Ser (GJA8), and Pro197Ser (GJA3), are predicted to be in the second extracellular domain of the respective connexin proteins. CONCLUSIONS: Our report extends the mutation spectrum of connexin genes GJA8 and GJA3 and confirms that connexin genes are among the most frequently mutated genes in hereditary cataracts. Our results suggest that connexin gene (GJA8 and GJA3) mutations occur in approximately 10% (4/40 families) of families with congenital hereditary cataracts in a population from southern India.

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.

Mapping of locus for autosomal dominant retinitis pigmentosa on chromosome 6q23.

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal degenerative disorders resulting in severe visual loss and blindness that have remained incurable till date. We report the mapping of the disease locus in a 3-generation family of Indian origin with autosomal dominant RP (ADRP). Diagnosis of RP and recruitment was made after a complete clinical evaluation of all members. Manifestations of the disease included night blindness with blurred central vision in some cases, loss of peripheral vision, and diffuse degeneration of the retinal pigment epithelium. Linkage analysis using microsatellite markers was carried out on 34 members (14 affected). After testing for linkage to known retinal dystrophy loci as well as a subsequent genome-wide analysis, we detected linkage to markers on chromosome 6q23: D6S262 at 130 cM, D6S457 (130 cM) and D6S1656 (131 cM) gave significant 2-point LOD scores of 3.0-3.8. Multipoint LOD scores of ≥3.0 were obtained for markers between 121 and 130 cM. Haplotype analysis with several markers in the same region on chromosome 6 shows a disease-cosegregating region of about 25 Mb between 109 and 135 Mb. There are no known RP genes in this interval, which contains >100 genes. This study provides evidence for a novel ADRP locus on chromosome 6q23.

Mutations in TULP1, NR2E3, and MFRP genes in Indian families with autosomal recessive retinitis pigmentosa.

PURPOSE: To identify genes underlying autosomal recessive retinitis pigmentosa (ARRP) by homozygosity mapping. METHODS: Families with ARRP were recruited after complete ophthalmic evaluation of all members and diagnosis of RP by predefined criteria. Genomic DNA from affected members of 26 families was genotyped on Illumina single nucleotide polymorphism (SNP) 6.0 K arrays with standard procedures. Genotypes were evaluated for homozygous regions that were common and concordant between affected members of each family. The genes mapping to homozygous intervals within these families were screened for pathogenic changes with PCR amplification and sequencing of coding regions. Co-segegration of sequence changes with disease was determined within each pedigree, and each variation was tested for presence in 100 unrelated normal controls. RESULTS: A genome-wide scan for homozygosity showed homozygous regions harboring the tubby like protein 1 gene (TULP1; chromosome 6) in one family, the nuclear receptor subfamily 2, group E, member 3 gene (NR2E3; chromosome 15) in three families, and the membrane frizzled-related protein gene (MFRP; chromosome 11) in one family. Screening of the three genes in the respective families revealed homozygous disease-causing mutations in three families. These included a missense mutation in TULP1, a deletion-cum-insertion in NR2E3, and a single base deletion in MFRP. Patients from all three families had a rod-cone type of dystrophy with night blindness initially. The NR2E3 and MFRP genes were associated with fundus features atypical of RP. CONCLUSIONS: This study shows involvement of the TULP1, NR2E3, and MFRP genes in ARRP in Indian cases. Genome-wide screening with SNP arrays followed by a prioritized candidate gene evaluation is useful in identifying genes in these patients.

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 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.

Posterior microphthalmos pigmentary retinopathy syndrome.

Posterior Microphthalmos Pigmentary Retinopathy Syndrome (PMPRS). Posterior microphthalmos (PM) is a relatively infrequent type of microphthalmos where posterior segment is predominantly affected with normal anterior segment measurements. Herein, we report two siblings with posterior microphthalmos retinopathy syndrome with postulated autosomal recessive mode of inheritance. A 13-year-old child had PM and retinitis pigmentosa (RP) and his 7-year-old sister had PM, RP, and foveoschisis. The genetics of this syndrome and variable phenotype is discussed. Importance of being aware of posterior microphthalmos and its posterior segment associations is highlighted.

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.