Owen M. Siggs

Description:Axenfeld-Rieger Syndrome (ARS) is an autosomal dominant condition with both ocular and non-ocular manifestations. ARS is primarily caused by coding variants at the PITX2 or FOXC1 loci, yet many cases still remain undiagnosed. Here we used whole-genome sequencing to identify two non-coding structural variants associated with a typical presentation of PITX2 -associated ARS: one with a 450 kb deletion removing a series of conserved enhancer elements distal to PITX2 , and the second with a 12.5 Mb inversion displacing the PITX2 gene from these same enhancer elements. Neither variant disrupted the PITX2 gene itself, and therefore both were expected to reduce PITX2 expression by disrupting its proximity or access to enhancer elements. Enhancer-disrupting intergenic inversions therefore represent a unique genetic mechanism for the development of ARS, which should be carefully considered in the context of ARS and other conditions without a conclusive genetic diagnosis.

Description:Objective: Pseudoexfoliation syndrome (PEX) is a known risk factor for glaucoma, but its individual clinical course ranges from no glaucoma to total blindness. This study investigated whether polygenic risk scores (PRSs) built from variants collectively associated with open-angle glaucoma, intraocular pressure (IOP), and vertical cup-to-disc ratio (VCDR) can stratify individuals with pseudoexfoliation for the risk of glaucoma development. Methods: Retrospective multicohort study of 2 glaucoma registries and 1 population-based cohort. Methods: For the primary analysis, participants (n = 828) were classified as having PEX with glaucoma, PEX with suspected glaucoma, or PEX with no glaucoma. For the secondary analysis, a cohort of participants (n = 2460) were classified as having PEX with glaucoma, having PEX with no glaucoma, and being unaffected, and an independent cohort of participants (n = 3372) were classified as having primary open-angle glaucoma (POAG) or suspected POAG. Methods: Previously published and validated PRSs for open-angle glaucoma, IOP, and VCDR were expressed as a percentile, decile, or tertile of an ancestrally matched healthy population. Multivariable logistic and linear regressions and survival analyses were performed. Methods: The main outcome measures were odds of pseudoexfoliative glaucoma (PEX-G) and odds of clinically relevant outcomes. Results: Participants in the top tertile of the glaucoma PRS showed greater odds of receiving a PEX-G diagnosis (adjusted odds ratio [aOR], 4.22; 95% confidence interval [CI], 2.62-6.88; P < 0.001), greater odds of bilateral central vision loss (aOR, 3.43; 95% CI, 1.49-8.99; P = 0.007), and greater odds of bilateral incisional surgery (aOR, 3.35; 95% CI, 1.33-10.24; P = 0.018). Age at PEX-G diagnosis was 1 year younger with each increasing glaucoma PRS decile (1.06 years; 95% CI, 0.59-1.53 years; P < 0.001). Participants with manifest glaucoma and pseudoexfoliation showed a comparatively lower glaucoma PRS than counterparts with POAG. Conclusions: The PRSs for open-angle glaucoma, IOP, and VCDR stratify risk of glaucoma development and disease severity among individuals with PEX. Background: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Description:Background: Polygenic risk scores (PRS), which provide an individual probabilistic estimate of genetic susceptibility to develop a disease, have shown effective risk stratification for glaucoma onset. However, there is limited best practice evidence for reporting PRS and patient-friendly reports for communicating PRS effectively are lacking. Here we developed patient-centred PRS reports for glaucoma screening based on the literature, and evaluated them with participants using a qualitative research approach. Methods: We first reviewed existing PRS reports and literature on probabilistic risk communication. This informed the development of a draft glaucoma screening PRS report for a hypothetical high risk individual from the general population. We designed three versions of the report to illustrate risk using a pictograph, a pie chart and a bell curve. We then conducted semi-structured interviews to assess preference of visual risk communication aids, understanding of risk, content, format and structure of the reports. Participants were invited from an existing study, which aims to evaluate the clinical validity of glaucoma PRS among individuals > 50 years from the general population. Numeracy and literacy levels were assessed. Results: We interviewed 12 individuals. The cohort was highly educated (42% university education), all were European and 50% were female. Numeracy (mean 2.1 ± 0.9, range 0 to 3), graph literacy (mean 2.8 ± 0.8, range 0 to 4) and genetic literacy (mean 24.2 ± 6.2, range - 20 to + 46) showed a range of levels. We analysed the reports under three main themes: visual preferences, understanding risk and reports formatting. The visual component was deemed important to understanding risk, with the pictograph being the preferred visual risk representation, followed by the pie chart and the bell curve. Participants expressed preference for absolute risk in understanding risk, along with the written content explaining the results. The importance of follow-up recommendations and time to glaucoma onset were deemed important. Participants expressed varied opinions in the level of information and the colours used, which informed revisions of the report. Conclusions: Our study revealed preferences for reporting PRS information in the context of glaucoma screening, to support the development of clinical PRS reporting. Further research is needed to assess PRS communication in other groups representative of target populations and with other target audiences (e.g. referring clinicians), and its potential psychosocial impact in the wider community.

Description:Introduction: Primary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterized by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. We aimed to identify genetic and morphological variation that can be attributed to trabecular meshwork cell (TMC) dysfunction and raised IOP in POAG. Methods: 62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescence coupled with CellProfiler image analysis allowed for single-cell morphological profiling. Results: Many gene knockouts invoked DEGs relating to matrix metalloproteinases and interferon-induced proteins. We have prioritized genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified, suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, while knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. Conclusion: High-throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.