Genetic Studies in Primary Open Angle Glaucoma

Primary Open-Angle Glaucoma (POAG) is a progressive optic neuropathy characterised by retinal ganglion cell (RGC) degeneration, optic nerve head damage and visual field impairment, occurring in the presence of an open anterior chamber angle. Increasing age, elevated intraocular pressure (IOP) and family history are among the main risk factors for POAG. Familial risk provides evidence for a genetic contribution to the disease, with sibling relative risk estimated to be between 3 and 11. Mendelian family studies and genome-wide association studies (GWAS) have identified several glaucoma-associated genes and loci; however, these explain only a small proportion of the genetic contribution to POAG, leaving a large proportion of heritability unexplained. This thesis aimed to assess whether rare, coding and splice-region, variants in selected POAG-associated genes contribute to this unresolved genetic component. To do this, MYOC, the main gene associated with Mendelian forms of POAG, was first screened using PCR and Sanger sequencing, followed by targeted next-generation sequencing of selected candidate genes using a single-molecule Molecular Inversion Probe (smMIPs)–based strategy.

PCR and Sanger sequencing of six amplicons covering the coding regions and splice recognition signals of MYOC was performed in 219 patients with POAG. Pathogenic and potentially pathogenic MYOC variants were identified in five patients. Four carried the known premature stop-gain variant, p.(Q368*), one of whom also carried the missense change, p.(T419A), while one additional patient carried a frameshift variant p.(K484Rfs*9). To extend the genetic analysis, a multi-gene smMIPs panel targeting the coding regions and splice sites of 20 candidate glaucoma genes was designed and optimised across four Illumina MiSeq runs. The optimised smMIPs panel, consisting of 737 probes, was then used to screen genomic DNA from 272 POAG patients and 82 controls, on an Illumina NextSeq 2000. Overall, 95.5% of the probes achieved a mean coverage of at least 30×. Forty-three rare, coding or splice-site, variants with a population allele frequency below 1% were identified across 15 of the 20 genes in 49 of the 272 POAG patients (18.0%), compared with 13 rare variants in 15 of the 82 controls (18.3%). MYOC variants showed the clearest pathogenic Mendelian contribution to POAG, with variants detected in 6/272 patients (2.2%). Four rare TP53BP2 variants were also identified in 4/272 patients and prioritised as potentially pathogenic, including a frameshift variant p.(D951Efs*5), to be predicted to result in a truncated protein that may affect the p53-mediated apoptosis pathway that contributes to RGC loss. This result supports further investigation of TP53BP2 in POAG. The remaining rare variants were mostly observed in single individuals and remained of uncertain clinical significance.

This study successfully developed and applied an optimised smMIPs-based approach for targeted screening across glaucoma-associated genes. MYOC variants were confirmed to contribute to a small proportion of POAG cases, while TP53BP2 was identified as a candidate for further investigation. Overall, the findings support the complex genetic architecture of POAG, in which rare coding and splice-region variants in known and candidate genes explain only a subset of cases. Larger cohorts, family based segregation analysis and functional studies will be required to clarify the contribution of many of the remaining variants identified in this work.