title: Identifying glaucoma susceptibility genes in extended families
Creators: name: Graham, PS
Publisher: name: University of Tasmania
Year: 2020

description: Glaucoma encompasses a heterogenous group of eye diseases and is the leading cause of irreversible blindness worldwide. Death of retinal ganglion cells causes degeneration of the optic nerve with resultant loss in vision. Primary open-angle glaucoma (POAG) is the most common form of glaucoma. An increase in pressure inside the eye, the intraocular pressure (IOP), is the main risk factor for developing POAG. Pressure reducing medication and surgery are currently the only preventative measures, and means of slowing the progression of the disease, available to those at risk of POAG or already diagnosed with this disease. There is currently no cure for glaucoma. POAG is a highly heritable, complex disease. Genome-wide association studies (GWAS) have identified many common risk variants, each with small effect sizes, which may increase an individual’s susceptibility to developing this disease. However, these variants together only account for around 3% of the heritability of POAG. Rare variants have been proposed as a source of the missing heritability, and the most powerful way to enrich for rare variants is through family studies. Linkage studies have identified genes harbouring rare variants which cause the disease in around 10% of cases. It is proposed that other, unidentified rare variants with large effect sizes, may be important in POAG pathogenesis. The clinical intermediate traits of POAG that have been used for successful genetic studies include IOP, vertical cup to disc ratio (VCDR) and central corneal thickness (CCT). These highly heritable, quantitative traits are measurable in all individuals, regardless of their POAG disease status. IOP and VCDR are significantly genetically correlated with POAG disease status, making them ideal endophenotypes to use to identify variants and genes involved with POAG pathogenesis. CCT is not significantly genetically correlated with POAG, but is a well-recognised risk factor for the disease, with thinner corneas associated with an increased risk of developing POAG. Identifying genes important in corneal development still gives us the opportunity to better understand the risk of developing POAG. GWAS have been very successful in identifying variants associated with each of these clinical intermediate traits, however, only a small proportion of the overall heritability for each trait is accounted for by these variants. For this study, we hypothesised that rare genetic variants associated with heritable ocular clinical traits could be identified in extended pedigrees and would improve our understanding of the genetic susceptibility to POAG. The first aim of this study was to determine the contribution of published genetic loci associated with POAG and related traits, to the trait variance of the clinical phenotypes in five extended POAG enriched families. The next aim was to use linkage analysis to identify regions harbouring potential POAG related risk variants in these five families. The final aim was to use in silico tools to investigate genetic variants within the most significant peaks identified from the linkage analysis. This study utilised whole exome sequencing data from 249 members of five large POAG enriched families, with detailed clinical data including IOP, VCDR and CCT measurements, to perform family-based association and linkage analyses within a variance components framework. These five extended families provide the opportunity to identify rare variants involved in disease due to the potential for variants to be enriched by transmission from founder individuals to successive generations. To identify the previously reported POAG loci, Aim 1 of this study began with a comprehensive literature review. To determine whether these loci contributed to the variance of the clinical intermediate traits of the families in this study, a family-based association analysis was conducted on genetic variants within literature reported regions. Several variants within published linkage regions were significantly associated with each of the three traits; including a single rare variant within MST1 on chromosome 3 (p = 9.60x10\(^{-5}\)) and a haplotype within FLRT3 on chromosome 20 (p = 1.17x10\(^{-4}\) to p = 3.18x10\(^{-4}\)), which were associated with a large increase in IOP in the family members who carry them. FLRT3 lies in a previously mapped glaucoma linkage region, GLC1K, and is an ideal candidate gene for further study. To identify novel genes associated with POAG and its endophenotypes, Aim 2 focused on linkage analyses using whole exome sequencing data, which were conducted for the IOP and VCDR traits in the five families. These are both significantly heritable traits, with heritabilities calculated at 52% (p = 5.13x10\(^{-10}\)) for IOP and 41% (p = 9.0x10\(^{-7}\)) for VCDR in the families of this study. Identity by descent relationships between family members were calculated directly from the whole exome sequencing data using IBDLD, a program specifically designed for dense genotyping data. Variance components linkage analysis was conducted using SOLAR, including correction for both the ascertainment of the POAG enriched families used in this study, and non-normal trait distributions. Linkage peaks reaching significance were identified for IOP, but not VCDR. For IOP, peaks on chromosomes 9q34.3 and 15q11.2-13.2 reached full significance (maximum LOD = 4.25 and 3.40 respectively), and peaks on chromosomes 2q23.1-31.1, 3p13-12.1, 6q24.2-25.1 and 7p11.2-q11.23 reached suggestive significance (maximum LOD &gt; 1.86). All but one of the peaks were novel, with the chromosome 15 peak completely overlapping the previously identified GLC1I locus. Aim 3 involved analysing the identified linkage peaks using in silico strategies. Variants within each region were prioritised based on their family-based association results, their effect on reducing the linkage peak when included in the linkage model, and predicted in silico functional effects. Candidate genes proposed by these analyses were further investigated by a pathways analysis and were assessed for association in a large GWAS for IOP. PKP4 and FMNL2, on chromosome 2, and HERC2, on chromosome 15, are proposed as promising candidate genes associated with increased IOP in the families of this study. FMNL2 has already been associated with increased IOP and with POAG in recent GWAS. The identification of FMNL2 as a possible risk gene in this study is positive and validates the approach taken in using linkage analysis of extended pedigrees as it had already been reported as associated with IOP. HERC2 is a novel gene for IOP identified in our current study, and very recently the locus has been (independently) identified by GWAS. PKP4 is also a novel gene for IOP and POAG. PKP4 and HERC2 require further research in relevant ocular tissues to determine whether they are functionally important and whether they could affect IOP regulation. This is one of the first studies to conduct linkage analysis using whole exome sequencing data on intermediate traits of POAG. This research has identified three candidate genes which may be involved with IOP regulation. Currently as the only modifiable risk factor for POAG, IOP is an ideal target to develop more effective treatments than are currently available. This research is necessary to improve our understanding of IOP regulation and its involvement with POAG pathogenesis.