Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus.

Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

COVID-19: The regional impact of COVID-19 on the certification of vision impairment in Northern Ireland.

PURPOSE: In this paper we highlight the impact which the disruption of secondary care ophthalmic services, resulting from COVID-19, has had on Sight Impairment (SI) and Severe Sight Impairment (SSI) certification in Northern Ireland. METHODS: Regional data on SI and SSI certification in the period after the onset of the lockdown (19 March 2020-18 June 2020) were compared to the period immediately before lockdown (1 January 2020-18 March 2020) and to the same periods in 2019. Change documented was compared to post-lockdown reductions in primary and secondary ophthalmic care activity. RESULTS: In 2019, during the 3-month period (19 March 2019-18 June 2019), 115 individuals were certified as sight impaired (SI 36, SSI 75, unspecified 4). Of those certified, 65 were female, 49 male. Principal causes of certification were: Age-related macular degeneration (AMD) (N = 45), glaucoma (N = 20) and diabetic eye disease (DED) (N = 10). Mean VA, recorded from the better eye of those certified, was 0.96 LogMAR. In the 3 months following the onset of lockdown (19 March 2020-18 June 2020), only 37 individuals were certified (SI 6, SSI 31), 12 female and 25 male. AMD was the most frequent cause of sight impairment (N = 20). There were only two DED certifications and one due to glaucoma. Mean VA in the better eye of those certified was 1.15LogMAR. The numbers of CVI certifications completed following the introduction of COVID-19 lockdown fell by 68%, compared to the 2019 data. There was a significant reduction in the proportion of female certifications (p = 0.01), and in certifications due to glaucoma (p = 0.02). The proportion of those certified as SSI as opposed to SI in the period after the onset of lockdown rose from 68% in 2019 to 84% in 2020. The mean VA of those certified in the period after the onset of lockdown, when compared to those certified in the other three periods, was worse by between 0.21 and 0.19 LogMAR (p = 0.06). Reductions reflected change in overall primary and secondary ophthalmic care activity. CONCLUSIONS: It is inconceivable that COVID-19 has reduced the incidence of sight-threatening eye disease. We must therefore assume that a flood of newly presenting sight loss will present once the pandemic has passed. New presentations will include those who would normally have attended during the lockdown period, and patients who, had they accessed ophthalmic care at the appropriate time, would have been saved from severe levels of blindness. The implications of the predicted increase in demand for medical, social and low vision related services are huge.

Plasma level of lipocalin 2 is increased in neovascular age-related macular degeneration patients, particularly those with macular fibrosis.

BACKGROUND: Previously, we and others have reported higher populations of circulating neutrophils in patients with neovascular age-related macular degeneration (nAMD). Neutrophil gelatinase-associated lipocalin (NGAL, also known as lipocalin-2, LCN2), an important innate immune mediator, is known to be critically involved in sterile inflammation-mediated organ failure, fibrosis, cancer progression and retinal degeneration. This study investigated the plasma levels of LCN2, matrix metalloproteinase 9 (MMP9) and LCN2/MMP9 complex in different types of nAMD and examined whether the levels were related to patients' responsiveness to anti-VEGF therapy. RESULTS: One hundred and seventy-four nAMD patients, including 108 with choroidal neovascularisation (CNV), 32 with retinal angiomatous proliferation (RAP), 23 with polypoidal choroidal vasculopathy (PCV) and 11 unclassified patients, and 43 healthy controls were recruited to this case-control study. Fifty-eight nAMD patients had macular fibrosis and 110 patients did not. Out of the 174 nAMD patients, 80 patients responded completely, 90 responded partially, and 4 did not respond to the anti-VEGF therapy. The plasma levels of LCN2 in nAMD patients (181.46 ± 73.62 ng/ml) was significantly higher than that in healthy controls (152.24 ± 49.55 ng/ml, P = 0.047). However, the difference disappeared after adjusting for age. A positive correlation between plasma level of LCN2 and age was observed in nAMD patients (r = 0.29, P = 0.0002) but not in healthy controls. The plasma level of LCN2 was also positively correlated with circulating neutrophils in nAMD patients (r = 0.34, p = 0.0007) but not in healthy controls (r = 0.057, p = 0.77). No correlation was observed between age and circulating neutrophils. Further analysis of nAMD subtypes uncovered a significantly higher level of LCN2 in patients with macular fibrosis even after adjusting for age. No relationship was observed between plasma levels of LCN2 and patients' responsiveness to anti-VEGF therapy. The plasma levels of MMP9 and LCN2/MMP9 complex were comparable between nAMD and controls. CONCLUSIONS: Our results suggest that higher plasma levels of LCN2 in nAMD are related to ageing and increased population of circulating neutrophils. Our results also suggest that higher levels of LCN2 may increase the risk of macular fibrosis in nAMD.

Evaluation of long-term intravitreal anti-vascular endothelial growth factor injections on renal function in patients with and without diabetic kidney disease.

BACKGROUND: Administering anti-vascular endothelial growth factor (anti-VEGF) by intraocular injection has been shown to have a safe systemic profile. Nevertheless, incidents of acute kidney injury following anti-VEGF injection have been reported. We assessed the long-term effect of multiple intravitreal anti-VEGF injections on measures of renal function in patients with diabetes including rate of change of estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (ACR). METHODS: A retrospective review of patients receiving diabetic macular oedema (DMO) treatment was undertaken. Serum creatinine, ACR, number of intravitreal anti-VEGF injections and clinical characteristics were collected from electronic healthcare records (EHR). A co-efficient of eGFR and ACR change with time was calculated over a mean duration of 2.6 years. Regression modelling was used to assess variation in the number of anti-VEGF injections and change in eGFR and ACR. RESULTS: The EHR of 85 patients with DMO (59% male, 78% type 2 diabetes mellitus [T2DM]) were reviewed. On average, 26.8 intravitreal anti-VEGF injections were given per patient over a mean duration of 31 months. No association between increasing number of anti-VEGF injections and rate of eGFR decline (beta = 0.04, 95% confidence intervals [CI]: - 0.02, 0.09; p = 0.22) or ACR change over time (beta = 0.02, CI: - 0.19, 0.23; p = 0.86) was detected, following adjustment for hypertension, cerebrovascular disease, T2DM, and medications taken. CONCLUSION: Our data suggests regular long-term intravitreal VEGF inhibition does not significantly alter the rate of change in eGFR and/or ACR with increasing number of treatment injections.

Peripheral blood mononuclear cells from neovascular age-related macular degeneration patients produce higher levels of chemokines CCL2 (MCP-1) and CXCL8 (IL-8).

BACKGROUND: Infiltrating immune cells including monocytes/macrophages have been implicated in the pathogenesis of neovascular age-related macular degeneration (nAMD). The aim of this study was to investigate the cytokine and chemokine expression and secretion profile of peripheral blood mononuclear cells (PBMCs) from nAMD patients and the relationship between the cytokine/chemokine expression profile and clinical phenotype of nAMD, including macular fibrosis, macular atrophy or the responsiveness to anti-VEGF therapy. METHODS: One hundred sixty-one nAMD patients and 43 controls were enrolled in this study. nAMD patients were divided into subgroups based on the presence/absence of (1) macular atrophy, (2) macular fibrosis and (3) responsiveness to anti-VEGF therapy; 25-30 ml of peripheral blood were obtained from all participants and 5 ml were used for serum collection, and the remaining were used for PBMC isolation using density gradient centrifugation. Intracellular cytokine expressions by PBMCs following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation were examined using flow cytometry. Cytokine productions in lipopolysaccharides (LPS)-or 1% oxygen -treated PBMC were measured using cytometric bead array (CBA) assay. In addition, cytokine and chemokine levels in the serum were also measured by CBA assay. RESULTS: PBMCs from nAMD patients secreted higher levels of IL-8, CCL2 and VEGF, especially following LPS and 1% oxygen stimulation, than those from controls. 60~80% of IL-8 producing cells were CD11b+CD3- monocytes. The percentage of CD11b+CD3- IL-8+ was significantly increased in nAMD patients compared to controls. PBMCs from nAMD patients without macular fibrosis produced the highest levels of IL-8 and CCL2, whilst PBMCs from nAMD patients with macular atrophy produced highest levels of VEGF. In addition, PBMCs from patients who partially responded to anti-VEGF produced higher levels of IL-8 compared to the cells from complete responders. Interestingly, serum level of CCL2 was not increased in nAMD patients although there was a trend of increased IL-8 in nAMD patients. CONCLUSIONS: PBMCs, in particular monocytes, may contribute to CNV development in nAMD through secreting elevated levels of IL-8, CCL2 and VEGF after they are recruited to the macula. Apart from VEGF, IL-8 and CCL2 may be additional targets for nAMD management.

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration.

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

Higher plasma levels of complement C3a, C4a and C5a increase the risk of subretinal fibrosis in neovascular age-related macular degeneration: Complement activation in AMD.

BACKGROUND: The aim of this study was to investigate the plasma levels of complement C3a, C4a, and C5a in different types of neovascular age-related macular degeneration (nAMD) and whether the levels were related to patients' responsiveness to anti-VEGF therapy. RESULTS: Ninety-six nAMD patients (including 61 with choroidal neovascularisation (CNV), 17 with retinal angiomatous proliferation (RAP), 14 with polypoidal choroidal vasculopathy (PCV) and 4 unclassified patients) and 43 controls were recruited to this case-control study. Subretinal fibrosis was observed in 45 nAMD patients and was absent in 51 nAMD patients. In addition, the responsiveness to anti-VEGF (Lucentis) therapy was also evaluated in nAMD patients. Forty-four patients were complete responders, 48 were partially responders, and only 4 patients did not respond to the therapy. The plasma levels of C3a, C4a and C5a were significantly higher in nAMD patients compared to controls. Further analysis of nAMD subgroups showed that the levels of C3a, C4a and C5a were significantly increased in patients with CNV but not RAP and PCV. Significantly increased levels of C3a, C4a and C5a were also observed in nAMD patients with subretinal fibrosis but not in those without subretinal fibrosis. Higher levels of C3a were observed in nAMD patients who responded partially to anti-VEGF therapy. CONCLUSIONS: Our results suggest increased systemic complement activation in nAMD patients with CNV but not RAP and PCV. Our results also suggest that higher levels of systemic complement activation may increase the risk of subretinal fibrosis in nAMD patients.

Next-generation sequencing-based molecular diagnosis of 82 retinitis pigmentosa probands from Northern Ireland.

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive photoreceptor degeneration. An accurate molecular diagnosis is essential for disease characterization and clinical prognoses. A retinal capture panel that enriches 186 known retinal disease genes, including 55 known RP genes, was developed. Targeted next-generation sequencing was performed for a cohort of 82 unrelated RP cases from Northern Ireland, including 46 simplex cases and 36 familial cases. Disease-causing mutations were identified in 49 probands, including 28 simplex cases and 21 familial cases, achieving a solving rate of 60 %. In total, 65 pathogenic mutations were found, and 29 of these were novel. Interestingly, the molecular information of 12 probands was neither consistent with their initial inheritance pattern nor clinical diagnosis. Further clinical reassessment resulted in a refinement of the clinical diagnosis in 11 patients. This is the first study to apply next-generation sequencing-based, comprehensive molecular diagnoses to a large number of RP probands from Northern Ireland. Our study shows that molecular information can aid clinical diagnosis, potentially changing treatment options, current family counseling and management.

Age-Related Macular Degeneration-Associated Genes in Alzheimer Disease.

OBJECTIVES: Given the clinical and pathological similarities between age-related macular degeneration (AMD) and Alzheimer disease (AD), to assess whether AMD-associated single nucleotide polymorphisms (SNPs), including those from complement-related genes, are associated with AD. DESIGN: A case-control association study-type design. SETTING: A UK tertiary care dementia clinic. PARTICIPANTS: 322 cognitively normal participants and 258 cases with a clinical diagnosis of AD. MEASUREMENTS: Polymorphisms in the following genes were studied: CFH, ARMS2, C2/CFB, C3, CFI/PLA2G12a, SERPING1, TLR3, TLR4, CRP, APOE, and TOMM40. Haplotypes were analysed for CFH, TOMM40, and APOE. Univariate analysis was performed for each genetic change and case-comparator status, and then correction for multiple testing performed. RESULTS: The presence of an ε4 APOE allele was significantly associated with AD. No association was evident between CFH SNPs or haplotypes, or other AMD-associated SNPs tested, and AD. The exceptions were TOMM40 SNPs, which were associated with AD even after correction for multiple comparisons. The associations disappeared, however, when entered into a regression model including APOE genotypes. CONCLUSIONS: The results for most SNPs tested, as well as CFH haplotypes, are novel. The functional effects of abnormal complement activity in AD's pathogenesis may be contradictory, but methodological reasons may underlie the lack of association-for example, genetic changes other than SNPs being involved.

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements.

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Retinal and Choroidal Thickness in an Indigenous Population from Ghana: Comparison with Individuals with European or African Ancestry.

Purpose: To evaluate the thickness of the macular retina and central choroid in an indigenous population from Ghana, Africa and to compare them with those measured among individuals with European or African ancestry. Design: Cross-sectional study, systematic review, and meta-analyses. Participants: Forty-two healthy Ghanaians, 37 healthy individuals with European ancestry, and an additional 1427 healthy subjects with African ancestry from previously published studies. Methods: Macular retinal thickness in the fovea, parafovea, and perifovea and central choroidal thickness were extracted from OCT volume scans. Associations with ethnicity, age, and sex were assessed using mixed-effect regression models. Monte Carlo simulations were performed to determine the sensitivity of significant associations to additional potential confounders. Pooled estimates of retinal thickness among other groups with African ancestry were generated through systematic review and meta-analyses. Main Outcome Measures: Macular retinal thickness and central choroidal thickness and their association with ethnicity, age, and sex. Results: When adjusted for age and sex, the macular retina and central choroid of Ghanaians are significantly thinner as compared with subjects with European ancestry (P < 0.001). A reduction in retinal and choroidal thickness is observed with age, although this effect is independent of ethnicity. Meta-analyses indicate that retinal thickness among Ghanaians differs markedly from that of African Americans and other previously reported indigenous African populations. Conclusions: The thickness of the retina among Ghanaians differs not only from those measured among individuals with European ancestry, but also from those obtained from African Americans. Normative retinal and choroidal parameters determined among individuals with African or European ancestry may not be sufficient to describe indigenous African populations. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Vitreomacular interface abnormalities in the Ghanaian African.

BACKGROUND/OBJECTIVE: Describe vitreomacular interface abnormalities (VMIA) using spectral-domain optical coherence tomography (SD-OCT), and correlations with age-related macular degeneration (AMD) grade in Ghanaian Africans. SUBJECTS/METHODS: Prospective, cross-sectional study of adults aged ≥50 years recruited in Ghana AMD Study. Participant demographics, medical histories, ophthalmic examination, digital colour fundus photography (CFP) were obtained. High-resolution five-line raster OCT, Macular Cube 512 × 128 scans, and additional line scans in areas of clinical abnormality, were acquired. SD-OCT VMI features classified by International Vitreomacular Traction Study Group system and relationships to AMD grade were evaluated. OUTCOMES: VMIA prevalence, posterior vitreous detachment (PVD), vitreomacular adhesions (VMA), vitreomacular traction (VMT), epiretinal membranes (ERM), correlations with AMD grade. RESULTS: The full Ghana AMD cohort included 718 participants; 624 participants (1248 eyes) aged ≥50 years (range = 50-101, mean = 68.8), 68.9% female were included in this analysis. CFP with OCT scans were available for 776 eyes (397 participants); 707 (91.1%) had gradable CFP and OCT scans for both AMD and VMI grading forming the dataset for this report. PVD was absent in 504 (71.3%); partial and complete PVD occurred in 16.7% and 12.0% respectively. PVD did not increase with age (p = 0.720). VMIA without traction and macular holes were observed in 12.2% of eyes; 87.8% had no abnormalities. VMIA was not significantly correlated with AMD grade (p = 0.819). CONCLUSIONS: This provides the first assessment of VMIA in Ghanaian Africans. VMIA are common in Africans; PVD may be less common than in Caucasians. There was no significant association of AMD grade with VMIA.

Dual pathway inhibition with faricimab for previously treated neovascular age-related macular degeneration and diabetic macular oedema: guidance from a UK panel of retina specialists.

BACKGROUND/OBJECTIVES: Some eyes with neovascular age-related macular degeneration (nAMD) and centre-involving diabetic macular oedema (DMO) fail to respond sufficiently or lose response over time to standard of care intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapy. This paper explores clinical scenarios for switching to dual action angiopoietin-2 (Ang-2)/VEGF-A inhibitor faricimab (Vabysmo, Roche Products Limited) in previously anti-VEGF-treated patients. METHODS: A national steering group meeting of UK retina specialists was held in London on 27 October 2023. Clinician practice and experience were reviewed together with pivotal clinical trial data and early findings from real-world settings. Roche Products Limited facilitated and funded the meeting. RESULTS: While there is no standardised protocol for identifying suboptimal response, the authors review relevant clinical biomarkers of disease activity used in routine clinical practice to determine patient response and guide treatment decisions. Common reasons identified for considering a change of treatment were lack of efficacy demonstrated by suboptimal anatomic or visual improvement and insufficient durability of response. The panel outline strategies for switching to faricimab among eligible patients with a prior anti-VEGF treatment history, with initial monthly loading doses or maintaining the previous treatment interval before attempting to extend, that may be integrated into current treat-and-extend (T&E) clinical pathways for treating patients with nAMD and DMO. General considerations for switching between treatments are also reviewed. CONCLUSION: Clinicians may consider a treatment switch to faricimab in nAMD and DMO patients who have suboptimal disease control or insufficient durability of response on initial anti-VEGF therapy.

Investigation of genetic variation in scavenger receptor class B, member 1 (SCARB1) and association with serum carotenoids.

OBJECTIVE: To investigate association of scavenger receptor class B, member 1 (SCARB1) genetic variants with serum carotenoid levels of lutein (L) and zeaxanthin (Z) and macular pigment optical density (MPOD). DESIGN: A cross-sectional study of healthy adults aged 20 to 70. PARTICIPANTS: We recruited 302 participants after local advertisement. METHODS: We measured MPOD by customized heterochromatic flicker photometry. Fasting blood samples were taken for serum L and Z measurement by high-performance liquid chromatography and lipoprotein analysis by spectrophotometric assay. Forty-seven single nucleotide polymorphisms (SNPs) across SCARB1 were genotyped using Sequenom technology. Association analyses were performed using PLINK to compare allele and haplotype means, with adjustment for potential confounding and correction for multiple comparisons by permutation testing. Replication analysis was performed in the TwinsUK and Carotenoids in Age-Related Eye Disease Study (CAREDS) cohorts. MAIN OUTCOME MEASURES: Odds ratios for MPOD area, serum L and Z concentrations associated with genetic variations in SCARB1 and interactions between SCARB1 and gender. RESULTS: After multiple regression analysis with adjustment for age, body mass index, gender, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, smoking, and dietary L and Z levels, 5 SNPs were significantly associated with serum L concentration and 1 SNP with MPOD (P<0.01). Only the association between rs11057841 and serum L withstood correction for multiple comparisons by permutation testing (P<0.01) and replicated in the TwinsUK cohort (P = 0.014). Independent replication was also observed in the CAREDS cohort with rs10846744 (P = 2×10(-4)), an SNP in high linkage disequilibrium with rs11057841 (r(2) = 0.93). No interactions by gender were found. Haplotype analysis revealed no stronger association than obtained with single SNP analyses. CONCLUSIONS: Our study has identified association between rs11057841 and serum L concentration (24% increase per T allele) in healthy subjects, independent of potential confounding factors. Our data supports further evaluation of the role for SCARB1 in the transport of macular pigment and the possible modulation of age-related macular degeneration risk through combating the effects of oxidative stress within the retina. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosures may be found after the references.

Usher Syndrome on the Island of Ireland: A Genotype-Phenotype Review.

Purpose: Usher syndrome (USH) is a genetically heterogeneous group of autosomal recessive (AR) syndromic inherited retinal degenerations (IRDs) representing 50% of deaf-blindness. All subtypes include retinitis pigmentosa, sensorineural hearing loss, and vestibular abnormalities. Thorough phenotyping may facilitate genetic diagnosis and intervention. Here we report the clinical/genetic features of an Irish USH cohort. Methods: USH patients were selected from the Irish IRD registry (Target 5000). Patients were examined clinically (deep-phenotyping) and genetically using a 254 IRD-associated gene target capture sequencing panel, USH2A exon, and whole genome sequencing. Results: The study identified 145 patients (24.1% USH1 [n = 35], 73.8% USH2 [n = 107], 1.4% USH3 [n = 2], and 0.7% USH4 [n = 1]). A genetic diagnosis was reached in 82.1%, the majority (80.7%) being MYO7A or USH2A genotypes. Mean visual acuity and visual field (VF) were 0.47 ± 0.58 LogMAR and 31.3° ± 32.8°, respectively, at a mean age of 43 years. Legal blindness criteria were met in 40.7%. Cataract was present in 77.4%. ADGRV1 genotypes had the most VF loss, whereas USH2A patients had greater myopia and CDH23 had the most astigmatism. Variants absent from gnomAD non-Finnish Europeans and ClinVar represented more than 20% of the variants identified and were detected in ADGRV1, ARSG, CDH23, MYO7A, and USH2A. Conclusions: USH is a genetically diverse group of AR IRDs that have a profound impact on affected individuals and their families. The prevalence and phenotype/genotype characteristics of USH in Ireland have, as yet, gone unreported. Understanding the genotype of Irish USH patients may guide clinical and genetic characterization facilitating access to existing/novel therapeutics.

Detailed analysis of an enriched deep intronic ABCA4 variant in Irish Stargardt disease patients.

Over 15% of probands in a large cohort of more than 1500 inherited retinal degeneration patients present with a clinical diagnosis of Stargardt disease (STGD1), a recessive form of macular dystrophy caused by biallelic variants in the ABCA4 gene. Participants were clinically examined and underwent either target capture sequencing of the exons and some pathogenic intronic regions of ABCA4, sequencing of the entire ABCA4 gene or whole genome sequencing. ABCA4 c.4539 + 2028C > T, p.[= ,Arg1514Leufs*36] is a pathogenic deep intronic variant that results in a retina-specific 345-nucleotide pseudoexon inclusion. Through analysis of the Irish STGD1 cohort, 25 individuals across 18 pedigrees harbour ABCA4 c.4539 + 2028C > T and another pathogenic variant. This includes, to the best of our knowledge, the only two homozygous patients identified to date. This provides important evidence of variant pathogenicity for this deep intronic variant, highlighting the value of homozygotes for variant interpretation. 15 other heterozygous incidents of this variant in patients have been reported globally, indicating significant enrichment in the Irish population. We provide detailed genetic and clinical characterization of these patients, illustrating that ABCA4 c.4539 + 2028C > T is a variant of mild to intermediate severity. These results have important implications for unresolved STGD1 patients globally with approximately 10% of the population in some western countries claiming Irish heritage. This study exemplifies that detection and characterization of founder variants is a diagnostic imperative.

The association between complement component 2/complement factor B polymorphisms and age-related macular degeneration: a HuGE review and meta-analysis.

The authors performed a systematic review of the association of complement component 2(C2)/complement factor B (CFB) gene polymorphisms with age-related macular degeneration (AMD). In total, data from 19 studies published between 2006 and 2011 were pooled for 4 polymorphisms: rs9332739 and rs547154 in the C2 gene and rs4151667 and rs641153 in the CFB gene. Data extraction and assessments for risk of bias were independently performed by 2 reviewers. Allele frequencies and allele and genotypic effects were pooled. Heterogeneity and publication bias were explored. Pooled minor allele frequencies for all 4 SNPs were between 4.7% and 9.6% for all polymorphisms, except for an Indian population in which the C allele at rs9332739 was the major allele. For the C2 polymorphisms, the minor C allele at rs9332739 and the minor T allele at rs547154 carried estimated relative risks (odds ratios) of 0.55 (95% confidence interval (CI): 0.46, 0.65) and 0.47 (95% CI: 0.39, 0.57), respectively. For the CFB polymorphisms, the minor A alleles at rs4151667 and rs614153 carried estimated risks of 0.54 (95% CI: 0.45, 0.64) and 0.41 (95% CI: 0.34, 0.51), respectively. These allele effects contributed to an absolute lowering of the risk of all AMD in Caucasian populations by 2.0%-6.0%. This meta-analysis provides a robust estimate of the protective association of C2/CFB with AMD.

Systems biology-based analysis implicates a novel role for vitamin D metabolism in the pathogenesis of age-related macular degeneration.

Vitamin D has been shown to have anti-angiogenic properties and to play a protective role in several types of cancer, including breast, prostate and cutaneous melanoma. Similarly, vitamin D levels have been shown to be protective for risk of a number of conditions, including cardiovascular disease and chronic kidney disease, as well as numerous autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases and type 1 diabetes mellitus. A study performed by Parekh et al. was the first to suggest a role for vitamin D in age-related macular degeneration (AMD) and showed a correlation between reduced serum vitamin D levels and risk for early AMD. Based on this study and the protective role of vitamin D in diseases with similar pathophysiology to AMD, we examined the role of vitamin D in a family-based cohort of 481 sibling pairs. Using extremely phenotypically discordant sibling pairs, initially we evaluated the association of neovascular AMD and vitamin D/sunlight-related epidemiological factors. After controlling for established AMD risk factors, including polymorphisms of the genes encoding complement factor H (CFH) and age-related maculopathy susceptibility 2/HtrA serine peptidase (ARMS2/HTRA1), and smoking history, we found that ultraviolet irradiance was protective for the development of neovascular AMD (p = 0.001). Although evaluation of serum vitamin D levels (25-hydroxyvitamin D [25(OH)D]) was higher in unaffected individuals than in their affected siblings, this finding did not reach statistical significance. Based on the relationship between ultraviolet irradiance and vitamin D production, we employed a candidate gene approach for evaluating common variation in key vitamin D pathway genes (the genes encoding the vitamin D receptor [VDR]; cytochrome P450, family 27, subfamily B, polypeptide 1 [CYP27B1]; cytochrome P450, family 24, subfamily A, polypeptide 1 [CYP24A1]; and CYP27A1) in this same family-based cohort. Initial findings were then validated and replicated in the extended family cohort, an unrelated case-control cohort from central Greece and a prospective nested case-control population from the Nurse's Health Study and Health Professionals Follow-Up Studies, which included patients with all subtypes of AMD for a total of 2,528 individuals. Single point variants in CYP24A1 (the gene encoding the catabolising enzyme of the vitamin D pathway) were demonstrated to influence AMD risk after controlling for smoking history, sex and age in all populations, both separately and, more importantly, in a meta-analysis. This is the first report demonstrating a genetic association between vitamin D metabolism and AMD risk. These findings were also supplemented with expression data from human donor eyes and human retinal cell lines. These data not only extend previous biological studies in the AMD field, but further emphasise common antecedents between several disorders with an inflammatory/immunogenic component such as cardiovascular disease, cancer and AMD.

Molecular diagnosis for heterogeneous genetic diseases with targeted high-throughput DNA sequencing applied to retinitis pigmentosa.

BACKGROUND: The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa. METHODS: A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls. RESULTS AND CONCLUSIONS: Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A→T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Surgical planning during a pandemic: Identifying patients at high risk of severe disease or death due to COVID-19 in a cohort of patients on a cataract surgery waiting list.

BACKGROUND: The delivery of cataract surgery during the COVID-19 pandemic is challenging because of the risk of nosocomial SARS-CoV-2 infection when patients attend hospital for elective care. In order to ascertain the risk to patients awaiting cataract surgery, this study aimed to identify the presence of systemic comorbidities that are associated with a high risk of severe disease or death due to COVID-19. METHODS: A prospective study of 315 patients (630 eyes) was conducted from 3rd June to 31st July 2020. An electronic health record was used to identify any systemic comorbidities that would render a patient 'clinically extremely vulnerable' to COVID-19, as outlined by the Department of Health for Northern Ireland. Patient demographics, best-corrected visual acuity (VA) and risk of postoperative anisometropia were also recorded. RESULTS: The median age of patients awaiting cataract surgery was 76 years (range 22-97). Of the 315 patients, 72% were aged over 70 and 16% were aged over 85. A systemic comorbidity that would confer high risk status was identified in 21% of patients. This high risk status was attributable to severe respiratory disease, cancer, and immunosuppression therapies in the majority of cases. The high risk group were younger than those deemed non-high risk, but there were no significant differences with respect to gender, anticipated degree of surgical difficulty, VA, or whether the patient was undergoing first or second eye surgery. Of those patients awaiting first eye cataract surgery, the mean VA in the listed eye was 0.84 logMAR and 39% (70/179) had a VA <0.3 logMAR (6/12 Snellen acuity) in their fellow eye. 57% of patients were awaiting first eye surgery, and 32% of those patients would be at risk of symptomatic anisometropia postoperatively. CONCLUSION: One-fifth of patients awaiting cataract surgery were found to be at high risk of severe disease or death from COVID-19 and these patients may experience delays in their surgical care. Additional planning is required in order to minimise the morbidity associated with delayed cataract surgery.