Pieter Bonnemaijer

Glaucoma is the most important cause of irreversible blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype of glaucoma, and the least understood. Intra-ocular pressure (IOP), family history, and race are well-known risk factors. Persons from African ancestry have a 3-5x increased risk of POAG; they have a more severe course of disease with a higher risk of blindness. Given the familial nature and racial predisposition genome-wide association studies (GWAS) have identified many genetic risk factors for POAG and POAG endophenotypes, but these factors explain only a minor proportion of POAG cases. Strikingly, these studies are predominantly based on study participants of European descent. It is likely that gene finding will be more effective in study populations which have a high frequency of disease and a more severe phenotype. Chapter 1 gives a general introduction and describes the aims of the thesis.

The aims of this thesis were:
1. to address differences in POAG phenotype in sub-Saharan African populations compared to European populations
2. to identify genetic variants associated with POAG in sub-Saharan African populations
3. to apply novel approaches to study genetic and phenotypic associations with glaucoma endophenotypes.

Phenotypic variation of POAG in sub-Saharan African and European populations
Chapter 2.1 discusses difference in POAG presentation across Tanzanian, South African Black, South African Coloured and a white Dutch population. We showed that Black sub-Saharan Africans (SSA) glaucoma patients presented with a much more severe and aggressive phenotype compared to white Dutch patients. In particular the IOP at presentation was 5-7 mm Hg higher compared to the Dutch and visual acuity was already severely affected at time of presentation. The Cape Coloured population had an intermediate phenotype, but more similar to the Dutch. SSA black patients presented also at much younger age, this indicates that the disease is more progressive and destructive in this population. We therefore emphasize that programs to create disease awareness are of particular importance in Africa.

In Chapter 2.2 we aimed to further elucidate the relationships between genetic ancestry and central corneal thickness (CCT) and IOP. While some studies have pointed towards CCT as an independent risk factor for POAG, irrespective of its bias on Goldmann IOP measurements. Review of African literature could not validate this concept in case-control studies. In a highly admixed group of Coloured glaucoma patients and controls we were able to study these relationships in an unbiased approach by measuring the compound of genetic African ancestry (GAA). We found that GAA was associated with thinner CCT and that increase in GAA in POAG patients was associated with higher IOP. A key point was that the difference in CCT between POAG patients and controls was reduced at higher proportions of GAA.

Novel genetic variants associated with POAG in populations from Sub-Sahara African ancestry
In Chapter 3.1 we showed the results from the first GWAS of POAG comprising participants from continental Africa. We identified a novel candidate locus encompassing EXOC4 reaching genome-wide significance in discovery stage. EXOC4 encodes a protein involved in exocytosis, an important process during outgrowth of neurons and synaptogenesis. Replication in various cohorts from SSA heritage failed, likely due to divergent LD patterns that characterize African populations giving rise to low statistical power. A genetic risk score considering previous identified POAG variants in European and Asian studies was statistically significant associated with POAG in our study. Though the individual effect sizes of the studied variants were substantially smaller compared to the European and Asian results. Our results therefore suggest that established genetic risk factors may be implicated in SSA POAG, but probably to a lesser extent. And so do not explain the higher disease load encountered in these populations.

To improve statistical power we joined an international genetic consortium of POAG studies comprising over 26 000 persons from SSA descent or SSA admixed heritage (The Genetics of Glaucoma in People of African Descent (GGLAD) Consortium). In Chapter 3.2 we present the results from a large multi-stage GWAS performed in this consortium. We found one genomic region reaching genome wide significance in the APBB2 gene, a gene that is involved in the proteolytic processing of amyloid precursor protein. This association is unique to African diaspora populations. The amyloid precursor protein is required for normal development of the retina. However, proteolytic processing produces amyloid beta peptides, which are toxic and aggregate to form amyloid plaques, a well-known hallmarks of Alzheimer’s disease. Explanatory immunohistochemical analysis of human retinal and primary visual cortex tissues from post-mortem African Americans suggested a potential relationship between the APBB2 risk allele, increased APBB2 expression in the retina, and associated increased β-amyloid plaque deposition. These proposed pathologic mechanisms require further investigation in larger samples.

Phenotypic and genetic associations with glaucoma endophenotypes
In Chapter 4.1 we investigated the systemic and ocular determinants of peripapillary retinal nerve fiber layer thickness (pRNFLT) in 8 population based studies of the E3 consortium. We confirmed previously reported associations of age and spherical equivalent with pRNFLT and identified several additional factors associated with pRNFLT. We found that IOP, stroke and hypertension were associated with thinner pRNFLT. Also, we found a trend in reduced pRNFLT in patients with dementia. Our results indicate that also non-glaucoma traits could affect pRNFLT measurements. Microvascular disease and neuronal degeneration should therefore be taken into account when interpreting the pRNFLT in glaucoma screening and management.

Chapter 4.2 examines novel statistical methods to analyze phenotypically and genetically correlated glaucoma endophenotypes jointly, and to find pleiotropic genetic loci. We performed a multi-trait GWAS of optic nerve head parameters (vertical cup-disc ratio, cup area and disc area), that showed high genetic and phenotypic correlation. We identified two genomic loci in PPPNRPS-PLEKHGP and near SERPINEP at genome-wide significance that replicate in independent Asian cohorts.

Finally, in Chapter 5 the main findings are placed in the context of my view on the pathophysiology of glaucoma and discusses the challenges encountered when conducting this research and proposes direction for future research.