Molecular Vision最新文献

Purpose: Retinitis pigmentosa (RP) is an inherited heterogeneous neurodegenerative retinal disease leading to blindness eventually. Currently, a large number of studies have explored its heterogeneity, but the genotype-phenotype correlation remains unclear. The present study aimed to explore genetic mutations and the correlation between genotype-phenotype in three RP families from the Chinese Han population.

Methods: Genomic DNA was obtained from peripheral blood samples of patients and their relatives and subjected to whole-exome and Sanger sequencing. The corresponding visual acuity and fundus examinations were also performed, including fundus photography and ophthalmologic examinations.

Results: In this study, three novel variants, including CERKL c.1482delT (p.Val495fs*), RPRH2 c.-5_3dup (p.Ala2Glufs*6), and RPGR c.1539del (p.Lys513Asnfs*), and a heterozygous mutation c.239-2A>G from three families were identified from three inheritance formats. All above variants were cosegregated, with the PRPH2 variant inherited in an autosomal dominant pattern, the CERKL variants in an autosomal recessive pattern, and the RPGR variant in an X-chromosome-linked recessive pattern, respectively.

Conclusions: This study laid the foundation for prenatal diagnosis of RP in three family pedigrees, offering a comprehensive understanding of the genetic and clinical characteristics of patients with RP, which provided theoretical support for addressing complex genetic heterogeneity to enable accurate prenatal screening and diagnosis, early detection, and treatment of RP.

Purpose: In the premolecular era, mammalian samples were embedded in epoxy resin blocks, such as Epon or Poly/Bed, for future evaluation by electron microscopy. However, use of these archival specimens for more modern mutation characterization studies can be challenging. The aim of this study was to determine if genomic DNA could be extracted from osmicated archival epoxy-embedded tissues to a quality suitable for short-amplicon PCR amplification.

Methods: We selected nine archived Epon, Araldite, or Poly/Bed embedded blocks of mammalian retinal and corneal tissue that were ~10 mm in length, embedded in the 1970s to 1990s, and had an extensive phenotypic description. Tissues were fixed in several combinations of glutaraldehyde and osmium before embedding. The blocks were shaved of excess resin, fragmented, and digested using an epoxy resin removal solution. The softened plastic was cut with a scalpel, washed, drained, and incubated at 56 °C overnight in a tissue lysis solution containing Proteinase K. Trizol was added to the samples, which were further mechanically homogenized. Chloroform was added, and the samples were centrifuged at 4 °C and 12,000 g. Upon phase separation, the upper clear phase was removed, 95% EtOH was added, the mix was filtered through a mini-genomic DNA extraction column and washed twice, and DNA was eluted with 10 mM Tris-HCL. Following final removal of phenol contamination using water-saturated ether, the purified DNA was quantified and used for PCR amplification.

Results: The extraction success was tested by targeted PCR amplification using primers that produced amplicons 90 to 260 bp in length and targeted genes relevant for inherited eye studies (progressive rod-cone degeneration-PRCD; rhodopsin-RHO; glucuronidase beta-GUSB1), plus an additional control gene receptor accessory protein 1 (REEP1). All but one of the epoxy-embedded eye samples were successfully amplified. Sanger sequencing confirmed the gene identity of amplified products.

Conclusions: By identifying methods to extract DNA from osmicated epoxy-embedded mammalian eye tissues, our results provide a valuable resource for determining the genetic basis of inherited diseases and for retroactively confirming molecular diagnoses based on microscopic analysis.

Purpose: To investigate the intraocular pressure (IOP) distribution of cynomolgus monkeys in different age groups after anesthesia and mydriasis.

Methods: A total of 158 young and 252 old cynomolgus monkeys, aged 3.95 (1.61; range, 1.38 to 5.97) and 18.80 (1.67; range, 15.21 to 24.69) years, respectively, were subjected to general anesthesia (intramuscular injection of Zoletil 4 mg/kg mixed with xylazine/ketamine 0.2 mg/kg). IOP was measured using an Icare tonometer at three time points: immediately following the onset of anesthesia, after stabilization but before mydriasis, and after mydriasis (induced by 1% tropicamide eye drops).

Results: No significant differences were observed between sexes or between eyes within the Macaca fascicularis colony. After anesthesia stabilization but before mydriasis, the IOP of the old M. fascicularis colony was 22.33 mm Hg (4.12; range, 12.33 to 37.33), whereas the IOP of the young M. fascicularis colony was 16.67 mm Hg (4.00; range, 10.67 to 25.67). After mydriasis, the IOP of the old M. fascicularis colony was 19.33 mm Hg (5.00; range 11.33 to 33.00), while the IOP of the young M. fascicularis colony was 15.67 mm Hg (4.50; range, 9.67 to 24.00). All measurements were conducted with the monkeys in a prone position. Notably, both the young and old colonies experienced a significant decrease in IOP after mydriasis (p < 0.001). Moreover, the reduction observed in the young colony was significantly greater than that in the old colony (p < 0.001).

Conclusions: The IOP levels of the old M. fascicularis colony were higher than those of the young one, regardless of whether it was after anesthesia, before mydriasis, or after mydriasis. Furthermore, monkeys of different age groups had different reductions in IOP under different interventions (anesthesia, mydriasis). Additionally, within the M. fascicularis colony, IOP was equivalent between males and females, as well as between the right and left eyes.

Purpose: Retinoblastoma, the most common eye tumor in children, can occur in hereditary or nonhereditary forms. In the hereditary form, various germline alterations, single nucleotide (SNVs) or copy number variations (CNVs) in the RB1 gene can be detected in patients. The aim of this study was to analyze cytogenetic germline changes in Polish patients with retinoblastoma and to assess whether cytogenetic techniques still have their application in diagnostics for retinoblastoma patients in the era of next-generation sequencing (NGS).

Methods: The results of genetic testing for germline mutations in patients with retinoblastoma performed between 2013 and 2023 were analyzed. In patients with cytogenetic alterations (CNV group, n = 19), the form of disease, age of onset, the first symptom, family history, and the type and extent of cytogenetic changes were verified. Comparative analyses were conducted between the CNV and SNV (n = 83) groups as well as the group of patients with normal genetic test results (n = 126).

Results: Cytogenetic changes were detected in 19 probands. These included: 16 deletions (10 partial and 6 whole gene deletions), 2 duplications, and 1 balanced translocation. Partial gene deletions included from 1 to 16 exons. In the CNV group, bilateral involvement predominated, with strabismus being the most common initial symptom. The mean age of onset was 16.9 months (median = 11 months; IQR, 8-22 months) and was lower in patients with bilateral involvement and partial gene deletions. Statistically significant differences compared to patients with normal genetic test results were observed in terms of laterality, the age of onset, initial symptom, and the family history of retinoblastoma. No such differences were found between the CNV and SNV groups.

Conclusions: Cytogenetic changes constitute a significant part of germline alterations in patients with retinoblastoma. Cytogenetic techniques should still be considered in diagnostic protocols, especially in patients with bilateral involvement and/or positive family history, as well as in parents of patients with CNV.

Bietti crystalline dystrophy (BCD), an autosomal recessive inherited retinal disorder caused by mutations in the CYP4V2 gene, has long remained therapeutically challenging. Recent advances in adeno-associated virus-based gene therapy have emerged as promising therapeutic strategies for patients with BCD. This review synthesizes current knowledge regarding the molecular genetic mechanisms underlying BCD pathogenesis and examines recent developments in diagnostic approaches and gene therapeutic interventions. We specifically analyze the clinical outcomes of three investigational gene therapy products-ZVS101e, NGGT001, and VGR-R01-focusing on their preliminary efficacy, safety profiles, and tolerability. Key parameters evaluated include dosing strategies, routes of administration, adverse event profiles, and improvements in best-corrected visual acuity. The collective evidence suggests these therapeutic candidates show potential for decelerating disease progression and enhancing visual function. Future optimization of these approaches should carefully consider administration sites and modalities, injection volumes, and disease severity at intervention. With gene replacement therapy for BCD advancing through late-stage clinical development, regulatory approval and clinical implementation may be anticipated in the near future.

Purpose: Cysteinyl leukotriene receptor 1 (CysLTR1), originally described as a proinflammatory G protein-coupled receptor, has been shown to possess diverse nonimmunological properties. One of these functions is to modulate glucose-stimulated insulin secretion in β cells. Furthermore, the inhibition of CysLTR1 increases retinal cell survival in early diabetic retinopathy. Nevertheless, the potential of CysLTR1 to modulate glucose levels in retinal vascular cells, such as endothelial cells (ECs) and pericytes (PCs), is unknown. Therefore, we determined the intracellular glucose levels in retinal cells in vitro after the inhibition of CysLTR1 under standard and high-glucose culture conditions.

Methods: Primary human ECs, PCs, and the ARPE-19 cell line were cultured under standard (5.5 mmol/l glucose + 27.5 mmol/l mannitol) and high-glucose (33.0 mmol/l) conditions in the absence and presence of the specific CysLTR1 antagonists montelukast and zafirlukast for 1, 3, and 7 days. CysLTR1 expression was determined by immunofluorescence microscopy. CysLT secretion was measured by enzyme-linked immunosorbent assay. The effects of high glucose and CysLTR1 inhibition on cell viability and intracellular glucose levels were analyzed by luminescence-based assays. Furthermore, the transendothelial and transepithelial electrical resistance of the ECs and ARPE-19 monolayers was measured.

Results: CysLTR1 inhibition under standard glucose culture conditions increased the cellular glucose levels in retinal ECs, PCs, and ARPE-19 cells after 1 and 3 days of treatment. Under high-glucose culture conditions, CysLTR1 inhibition for 1 day reduced the intracellular glucose level in ARPE-19 cells. However, CysLTR1 inhibition for 3 days increased the level of intracellular glucose in ARPE-19 cells under high-glucose culture conditions. Furthermore, CysLTR1 inhibition reduced the tightness of the EC and ARPE-19 monolayers under standard culture conditions but increased the tightness of the ARPE-19 monolayers under high-glucose conditions.

Conclusions: CysLTR1 is considered a potential target for the treatment of type 2 diabetes and early diabetic retinopathy. Our data revealed that CysLTR1 activity directly regulates cellular glucose levels in retinal cells, supporting these hypotheses. Interestingly, the effect of CysLTR1 activity on glucose levels was reversed under acute metabolic stress. Thus, the activity of CysLTR1 appears to be more complex in terms of glucose metabolism and needs to be studied in more detail.

Purpose: Hair anomalies represent a common associated symptom of congenital cataracts. Early diagnosis is crucial for treatment and predicting prognosis. However, the insidious and nonspecific nature of the symptoms in young children makes diagnosis challenging, often necessitating tools such as whole-exome sequencing (WES) for accurate assessment. This study aims to propose a simple and expedient approach to guide clinical management by analyzing phenotype-genotype correlations.

Methods: A prospective cohort study was conducted among participants who underwent clinical examinations and WES between 2021 and 2023. Bioinformatic analysis was performed. In total, 170 unrelated congenital cataract probands were tested. The suspected pathogenic variants were validated through Sanger sequencing in both the probands and available family members. Correlation analyses were then performed, integrating clinical characteristics, cataract phenotype, and genotype data.

Results: Nine probands presented with both cataracts and hair anomalies. Potential pathogenic variants were detected in all patients with hair anomalies, including a novel variant in LSS. Phenotype-genotype analysis supports the classification of patients into two groups: hypotrichosis 14 and ichthyosis follicularis with atrichia and photophobia syndrome 2, based on the cataract phenotype, severity of the hair anomalies, and the presence of corneal pannus. These patients should be monitored closely for the development and progression of glaucoma and corneal lesions.

Conclusions: We identified nine probands with hair anomalies in our large cohort of congenital cataract probands. Using WES and comprehensive clinical examinations, we established definitive diagnoses, broadened the phenotype and genotype, and proposed phenotype-genotype correlations.

Purpose: To characterize the aggregation behavior of the γD-crystallin protein in an acidic environment with a focus on the formation of intermediate species. The research employs fluorescence correlation spectroscopy to unravel the intricate molecular events leading to aggregation, contributing to a comprehensive understanding of cataract formation.

Methods: The kinetics of γD-crystallin protein aggregation were studied with a reversed-phase high-performance liquid chromatography sedimentation assay, a ThT binding assay, and light scattering. We used fluorescence correlation spectroscopy (FCS) to recognize intermediate aggregate species and characterized them with Fourier transform infrared spectroscopy (FTIR). Further, the morphologic characterization of aggregates was done by transmission electron microscopy (TEM), and their hydrophobic characteristics were analyzed using the 8-anilino-1-naphthalenesulfonic acid binding assay.

Results: A negligible lag phase was observed in the aggregation kinetic experiments of the γD-crystallin protein. Pentamer, 25-mer, and higher oligomer intermediates were formed on the aggregation pathway. Conformation studies by FCS and FTIR have shown that oligomers are rich in cross-β sheet and random coil structure; however, they constitute more α-helix and less cross-β sheet structure than fibrils. TEM analysis revealed the approximate size of oligomers (diameter ~10 nm), protofibrils (~15 nm), and fibrils (~15 to ~35 nm).

Conclusions: In this study, we reported the presence of various intermediate aggregate species formed on the aggregation pathway of γD-crystallin protein at low pH. This will open new areas of research in understanding the detailed aggregation mechanism and aggregation hotspot within unfolded γD-crystallin monomers. The insights gained will also pave the way for future research in the realm of amyloid formation in cataract.

Purpose: Cyclic nucleotide-gated (CNG) channels are ligand-gated ion channels that transduce light signals into electrical signals in the retinal photoreceptors. Pathogenic variants in CNG channel genes are reported to cause inherited retinal dystrophies (IRDs). The current study used targeted panel sequencing to describe the mutational spectrum of CNG channel genes in familial cases of IRDs from eight consanguineous Pakistani families.

Methods: The current study included consanguineous Pakistani families with at least two affected members. DNA was extracted from whole blood samples by the phenol-chloroform method. Two affected members from each family were initially analyzed using targeted panel sequencing of 344 known IRD genes. The pathogenicity of candidate variants was assessed using the American College of Medical Genetics and Genomics guidelines. Segregation testing was performed by Sanger sequencing.

Results: Results of eight IRD families revealed a total of four reported variants in CNGA3 (c.827A>G, c.955T>C, c.1641C>A, c.1810C>T) and three novel variants, including c.1633A>T, c.800G>T, and c.1153T>C in CNGA1, CNGA3, and CNGB3 genes, respectively, segregating in each respective family. Among disease-causing variants identified in our study cohort, 87.5% were missense. Furthermore, one of the reported missense variants (i.e., c.1641C>A in CNGA3) was segregating in two unrelated families. All identified variants were homozygous and segregated in an autosomal recessive form.

Conclusions: CNGA3 was the most frequently mutated gene in our study cohort. Only the c.1641C>A variant of CNGA3 was repeated in two families, showing genetic diversity. The identification of three novel pathogenic variants in CNG channel genes in the present study reaffirms the allelic and genetic heterogeneity of IRDs in the Pakistani population.