Role of Mitochondrial Mutations in Ocular Aggregopathy.

Background Mitochondria are essential cellular organelles that are responsible for oxidative stress-induced damage in age-dependent neurodegenerations such as glaucoma. Previous studies have linked mitochondrial DNA (mtDNA) mutations to cellular energy shortages that result in eye degeneration. Methodology To look for nucleotide variations in mtDNA in exfoliation syndrome/glaucoma (XFS/XFG), we performed a polymerase chain reaction (PCR) to amplify the entire coding region of the mitochondrial genome from peripheral blood of XFS/XFG (n = 25) patients and controls (n = 25). Results This study identified a total of 65 variations in XFS/XFG patients, of which 25 (38%) variations were non-synonymous single-nucleotide polymorphism (nsSNPs). Out of 25 nsSNPs, seven (five nsSNP in MT-ND4 and two in MT-ATP6 gene) were predicted as pathogenic using four different software, namely, SIFT, Polyphene2, mutation taster, and MutPred2. The pathogenic nsSNPs were then subjected to structural change analysis using online tools. Conclusions The pathogenic nsSNPs were found in both proteins' transmembrane domains and were expected to be conserved, but with lower protein stability (ΔΔG <- 0.5), indicating a possibly harmful effect in exfoliation. However, three-dimensional protein analysis indicated that the predicted mutations in MT-ND4 and MT-ATP6 were unlikely to alter the protein function.

Mitochondrial Genome Alterations, Cytochrome C Oxidase Activity, and Oxidative Stress: Implications in Primary Open-angle Glaucoma.

Aim: To evaluate mitochondrial genome alterations, cytochrome c oxidase (COX) activity, and oxidative stress in primary open-angle glaucoma (POAG). Methodology: Whole mitochondrial genome was screened in 75 POAG cases and 105 controls by polymerase chain reaction (PCR) sequencing. COX activity was measured from peripheral blood mononuclear cells (PBMCs). A protein modeling study was done to evaluate the impact of G222E variant on protein function. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-isoprostane (8-IP), and total antioxidant capacity (TAC) were also measured. Results: A total of 156 and 79 mitochondrial nucleotide variations were found in the cohort of 75 POAG patients and 105 controls, respectively. Ninety-four (60.26%) variations spanned the coding region, and 62 (39.74%) variations spanned noncoding regions (D-loop, 12SrRNA, and 16SrRNA) of mitochondrial genome in POAG patients. Out of 94 nucleotide changes in coding region, 68 (72.34%) were synonymous changes, 23 (24.46%) non-synonymous, and three (3.19%) were found in the region coding for transfer ribonucleic acid (tRNA). Three changes (p.E192K in ND1, p.L128Q in ND2, and p.G222E in COX2) were found to be pathogenic. Twenty-four (32.0%) patients were positive for either of these pathogenic mitochondrial deoxyribonucleic acid (mtDNA) nucleotide changes. Majority of cases (18.7%) had pathogenic mutation in COX2 gene. Patients who harbored pathogenic mtDNA change in COX2 gene had significantly lower levels of COX activity (p < 0.0001) and TAC (p = 0.004), and higher levels of 8-IP (p = 0.01) as compared to patients who did not harbor this mtDNA. G222E changed the electrostatic potential and adversely impacted protein function of COX2 by affecting nonpolar interactions with neighboring subunits. Conclusion: Pathogenic mtDNA mutations were present in POAG patients, which were associated with reduced COX activity and increased levels of oxidative stress. Clinical significance: POAG patients should be evaluated for mitochondrial mutations and oxidative stress and may be managed accordingly with antioxidant therapies. How to cite this article: Mohanty K, Mishra S, Dada R, et al. Mitochondrial Genome Alterations, Cytochrome C Oxidase Activity, and Oxidative Stress: Implications in Primary Open-angle Glaucoma. J Curr Glaucoma Pract 2022;16(3):158-165.

Mindfulness Meditation Reduces Intraocular Pressure, Lowers Stress Biomarkers and Modulates Gene Expression in Glaucoma: A Randomized Controlled Trial.

BACKGROUND: Reducing intraocular pressure (IOP) in primary open-angle glaucoma (POAG) is currently the only approach to prevent further optic nerve head damage. However, other mechanisms such as ischemia, oxidative stress, glutamate excitotoxicity, neurotrophin loss, inflammation/glial activation, and vascular dysregulation are not addressed. Because stress is a key risk factor affecting these mechanisms, we evaluated whether mindfulness-based stress reduction can lower IOP and normalize typical stress biomarkers. MATERIALS AND METHODS: In a prospective, randomized trial 90 POAG patients (180 eyes; age above 45 y) were assigned to a waitlist control or mindfulness meditation group which practiced daily for 21 days. We measured IOP (primary endpoint), quality of life (QOL), stress-related serum biomarkers [cortisol, ß-endorphins, IL6, TNF-α, brain-derived neurotrophic factor (BDNF), reactive oxygen species (ROS), total antioxidant capacity (TAC)], and whole genome expression. RESULTS: Between-group comparisons revealed significantly lowered IOP in meditators (OD: 18.8 to 12.7, OS 19.0 to 13.1 mm Hg) which correlated with significantly lowered stress-biomarker levels including cortisol (497.3 to 392.3 ng/mL), IL6 (2.8 to 1.5 ng/mL), TNF-α (57.1 to 45.4 pg/mL), ROS (1625 to 987 RLU/min/104 neutrophils), and elevated ß-endorphins (38.4 to 52.7 pg/mL), BDNF (56.1 to 83.9 ng/mL), and TAC (5.9 to 9.3) (all P<0.001). These changes correlated well with gene expression profiling. Meditators improved in QOL (P<0.05). CONCLUSIONS: A short course of mindfulness-based stress reduction by meditation in POAG, reduces IOP, improves QOL, normalizes stress biomarkers, and positively modifies gene expression. Mindfulness meditation can be recommended as adjunctive therapy for POAG.

Identification and genotype phenotype correlation of novel mutations in SIX6 gene in primary open angle glaucoma.

BACKGROUND: Recently SIX1 and SIX6 genes have been associated with primary open angle glaucoma (POAG). This study was planned to do mutation screening in SIX1 and SIX6 genes in North Indian POAG patients and correlate with clinical phenotypes. MATERIALS AND METHODS: SIX1 and SIX6 genes were amplified by PCR and sequenced in 115 POAG cases and 105 controls. Four pathogenecity prediction tools (MutationTaster, PolyPhen-2 HumDiv, PolyPhen-2 HumVar and SIFT) were used to predict the pathogenicity of the missense mutations. Protein modeling studies were done to predict the effect of the missense mutations on the protein structure and function. RESULTS: Two novel mutations p.R116G and p.R116E were observed in the SIX6 gene of patients with POAG. The mutations p.R116G and p.R116E were predicted to be pathogenic and replacement of R116 by G or E might lead to loss of interaction between DNA and R116 of wild type SIX6 protein. The patients with the mutation p.R116E had significantly more visual field damage (MD) and early age of onset of the disease. No sequence variations were observed in the SIX1 gene. CONCLUSION: These results expand the mutation spectrum of SIX6 gene and suggest that SIX6 gene plays an important role in POAG pathogenesis.

Oxidative DNA damage and reduced expression of DNA repair genes: Role in primary open angle glaucoma (POAG).

BACKGROUND: Controversy exists regarding the role of oxidative DNA damage and DNA repair in primary open angle glaucoma (POAG). We performed a case control study to test the hypothesis that oxidative DNA damage and base excision repair (BER) genes PARP1 and OGG1 are involved in POAG pathogenesis. MATERIALS AND METHODS: The study included 116 POAG patients and 116 cataract patients as controls. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were measured by ELISA. RNA was extracted from blood by Trizol and converted to cDNA. The relative quantification of PARP1 and OGG1 genes normalized to ß-actin was calculated by the 2-ΔCt method. Comparisons between groups were done by student's t-test and correlation between parameters was seen by Pearson correlation coefficient. All p values less than 0.05 were considered significant. RESULTS: Mean levels of 8-OHdG were (patients v/s controls) 19.53 ± 1.40 vs. 15.0 ± 2.6 ng/ml in plasma and 8.55 ± 1.94 vs. 5.15 ± 1.09 ng/ml in aqueous humor (p < 0.0001). Expression levels of PARP1 (0.44 ± 0.05 vs. 0.88 ± 0.04) and OGG1 (0.46 ± 0.05 vs. 0.8 ± 0.01) were significantly (p < 0.0001) less in the patients than controls. There was a significant negative correlation between the expression levels of PARP1 and OGG1 with plasma and aqueous 8-OHdG. There was a strong positive correlation between plasma and aqueous 8-OHdG levels. CONCLUSION: These results support our hypothesis that oxidative stress-induced DNA damage is associated with POAG. Increased oxidative DNA damage in POAG may be attributed to decreased expression of DNA repair enzymes of the BER pathway.

Neurodegenerative Eye Disorders: Role of Mitochondrial Dynamics and Genomics.

As a major source of cellular energy, mitochondria are critical for optimal ocular function. They are also essential for cell differentiation and survival. Mitochondrial mutations and oxidative damage to the mitochondrial DNA are important factors underlying the pathology of many ocular disorders. With increasing age, mitochondrial DNA damage accumulates and results in several eye diseases. It is evident that the mitochondrial genome is more susceptible to stress and damage than the nuclear genome, as it lacks histone protection, a nucleotide excision repair system, and recombination repair, and it is the source and target of free radicals. Accumulation of mitochondrial mutations beyond a certain threshold explains the marked variations in phenotypes seen in mitochondrial diseases and the molecular mechanisms related to the pathogenesis of several chronic disorders in the eye. This review details the structure and function of mitochondria and the mitochondrial genome along with the mitochondrial involvement in various neurodegenerative ophthalmic disorders.

A Unique Case of JOAG With Lamellar Ichthyosis With Rickets: A Case Report and Review of the Literature.

PURPOSE: Ichthyosis is known to have ocular associations such as blepharitis, hypertrophic conjunctivitis, corneal vascularization, ectropion, lagophthalmos, etc. However, no reports of its association with glaucoma are there, to the best of our knowledge. We report a unique case of juvenile open-angle glaucoma (JOAG) with lamellar ichthyosis. METHOD: A 16-year-old male child presented with a gradual, painless progressive diminution of vision in both eyes over a period of 3 years. Systemic examination revealed stunted body growth with knock-knees, suggestive of late-onset rickets. Generalized dry scaly lesions with erythema, along with hyperkeratosis of the palms and the soles, suggestive of lamellar ichthyosis were present. On ocular examination, the intraocular pressure was 36 mm Hg; optic nerve head examination revealed a horizontally oval disc with near total cupping in the right eye and total cupping in the left eye, with extensive neuroretinal rim thinning and pallor. Gonioscopy showed wide open angles with prominent iris processes. Screening of JOAG-associated genes (MYOC, NTF4, WDR36, and CYP1B1) and ichthyosis-associated gene (TGM1) was performed by the direct PCR-sequencing method. RESULTS: A diagnosis of JOAG with advanced glaucomatous optic neuropathy with lamellar ichthyosis and rickets was made. The patient underwent right followed by left eye trabeculectomy with 0.2 mg/dL MMC (for 1 min). Postoperatively, the intraocular pressure was 8 mm Hg at 1 week, and 12 to 14 mm Hg at the 6-week, the 3-month, and the 6-month follow-up, and the visual acuity was maintained in the right eye. No mutations in MYOC, NTF4, WDR36, CYP1B1, and TGM1 were observed in the patient and his family. CONCLUSIONS: An association of glaucoma with ichthyosis should be kept in mind. Therefore, a detailed baseline ocular examination in children with ichthyosis is required, as early detection of glaucoma could prevent irreversible blindness.

Screening of the LTBP2 gene in a north Indian population with primary congenital glaucoma.

PURPOSE: Primary congenital glaucoma (PCG), a severe form of glaucoma that presents early in life, is an autosomal recessive eye disorder that results from defects in anterior eye segment. Null mutations in LTBP2 were reported in patients with PCG in Pakistani and Iranian families. This study was aimed to identify the mutation profile of the LTBP2 gene in north Indian patients with PCG. METHODS: After ethical clearance, 54 unrelated patients with PCG who were either negative or heterozygous for MYOC, CYP1B1, and FOXC1 mutations and 50 ethnically matched non-glaucomatous controls were recruited for the study. PCG diagnosis was established by the presence of buphthalmos in at least one affected eye and associated high intraocular pressure before the age of 3 years. LTBP2 was screened in genomic blood DNA for mutations, with PCR and direct sequencing of PCR amplified fragments. RESULTS: We observed one intronic single nucleotide polymorphism (rs3742793) between exons 6 and 7 in the LTBP2 gene in 18 patients with PCG. This nucleotide change resulted in cytosine (C) being replaced by guanosine (G) at position g.75070493. No pathogenic variants were identified in the LTBP2 gene in our cohort of patients. CONCLUSIONS: LTBP2 gene mutations are not involved in the pathogenesis of primary congenital glaucoma in our patients. Thus, it is important to screen other glaucoma-associated loci and genes for involvement in congenital glaucoma in cases that are either negative or heterozygous for MYOC, CYP1B1, and FOXC1 mutations to have better insight into the disease pathogenesis.

Molecular Diagnostics and Genetic Counseling in Primary Congenital Glaucoma.

Primary congenital glaucoma (PCG) is a childhood irreversible blinding disorder with onset at birth or in the first year of life. It is characterized by the classical traid of symptoms viz. epiphora (excessive tearing), photophobia (hypersensitivity to light) and blepharospasm (inflammation of eyelids). The only anatomical defect seen in PCG is trabecular meshwork dysgenesis. PCG shows autosomal recessive mode of inheritance with considerable number of sporadic cases. The etiology of this disease has not been fully understood but some genes like CYP1B1, MYOC, FOXC1, LTBP2 have been implicated. Various chromosomal aberrations and mutations in mitochondrial genome have also been reported. Molecular biology has developed novel techniques in order to do genetic and biochemical characterization of many genetic disorders including PCG. Techniques like polymerase chain reaction, single strand conformational polymorphism and sequencing are already in use for diagnosis of PCG and other techniques like protein truncation testing and functional genomics are beginning to find their way into molecular workout of this disorder. In the light of its genetic etiology, it is important to develop methods for genetic counseling for the patients and their families so as to bring down its incidence. In this review, we ought to develop a genetic insight into PCG with possible use of molecular biology and functional genomics in understanding the disease etiology, pathogenesis, pathology and mechanism of inheritance. We will also discuss the possibilities and use of genetic counseling in this disease. How to cite this article: Faiq M, Mohanty K, Dada R, Dada T. Molecular Diagnostics and Genetic Counseling in Primary Congenital Glaucoma. J Current Glau Prac 2013;7(1):25-35.

Genetic, Biochemical and Clinical Insights into Primary Congenital Glaucoma.

Glaucoma is an irreversible form of optic neuropathy in which the optic nerve suffers damage in a characteristic manner with optic nerve cupping and retinal ganglion cell death. Primary congenital glaucoma (PCG) is an idiopathic irreversible childhood blinding disorder which manifests at birth or within the first year of life. PCG presents with a classical triad of symptoms (viz epiphora, photophobia and blepharospasm) though there are many additional symptoms, including large eye ball and hazy cornea. The only anatomical anomaly found in PCG is trabecular meshwork (TM) dysgenesis. PCG is an inheritable disease with established genetic etiology. It transmits through autosomal recessive mode. A number of cases are sporadic also. Mutations in many genes have been found to be causative in PCG and many are yet to be found. Mutations in cytochrome P4501B1 (CYP1B1) gene have been found to be the predominant cause of PCG. Other genes that have been implicated in PCG etiology are myocilin, Forkhead-related transcription factor C1 (FOXC1) and latent transforming growth factor beta-binding protein 2 (LTBP2). Mutations in these genes have been reported from many parts of the world. In addition to this, mitochondrial genome mutations are also thought to be involved in its pathogenesis. There appears to be some mechanism involving more than one genetic factor. In this review, we will discuss the various clinical, biochemical and genetic aspects of PCG. We emphasize that etiology of PCG does not lie in a single gene or genetic factor. Research needs to be oriented into a direction where gene-gene interactions, ocular embryology, ophthalmic metabolism and systemic oxidative status need to be studied in order to understand this disorder. We also accentuate the need for ophthalmic genetic facilities in all ophthalmology setups. How to cite this article: Faiq M, Sharma R, Dada R, Mohanty K, Saluja D, Dada T. Genetic, Biochemical and Clinical Insights into Primary Congenital Glaucoma. J Current Glau Prac 2013;7(2):66-84.