What is next for genetics and glaucoma?

Janey L. Wiggs

Twenty years ago the first of the glaucoma genetic linkage studies was completed and these have now yielded a handful of genes that cause early-onset familial glaucoma: MYOC (juvenile open-angle glaucoma), PITX2 (Axenfeld-Rieger syndrome), FOXC1 (anterior segment dysplasia and iris atrophy), PAX6 (Aniridia), CYP1B1 (primary congenital glaucoma), LTBP2 (primary congenital glaucoma), OPTN and TBK1 (familial normal-tension glaucoma).¹ In 2007, the first genome-wide association study (GWAS) for common complex forms of glaucoma was completed and subsequently this approach has yielded seven genes/genomic regions: LOXL1 (pseudoexfoliation syndrome), CAV1/CAV2 (POAG), CDKN2BAS (primary open-angle glaucoma (POAG) and NTG), SIX6 (POAG), TMCO1 (POAG), 8q22 (NTG), PLEKHA7 (angle closure), COL11A1 (angle closure).¹ Although these discoveries represent great progress, this collection of genes still only accounts for a fraction of glaucoma heritability. Future goals for glaucoma genetic research include finding more genes for early-onset familial glaucoma and adult-onset common glaucoma and using the genetic information to develop clinically meaningful gene-based screening, diagnostic and prognostic tests, as well as novel gene-based therapies.

Future goals for glaucoma genetic research include finding more genes for early-onset familial glaucoma and adult-onset common glaucoma.

Finding new glaucoma genes

Powerful new DNA sequencing technologies such as whole exome sequencing (WES) can be used to find disease-causing genes in smaller families and even individuals, eliminating the need for the very large families previously required for gene discovery using genetic linkage analysis. Identifying disease-causing genes using these older methods was laborious and time-consuming. WES examines the entire coding sequence (exome) of a patient and through comparison with the exomes from healthy controls or unaffected family members the DNA sequences unique to affected individuals can be identified. Genes with DNA sequence variants found only in affected individuals are likely to be the cause of the inherited disease. As the current collection of early-onset glaucoma genes only accounts for about 20% of affected families, WES is expected to find interesting novel genes in many more families in the near future. Showing that novel gene variants are indeed responsible for disease development remains a challenge. However, animal model systems such as morpholino knock down in zebrafish and disease modeling in iPS (induced pluripotent stem) cells are helpful.

The current collection of genes contributing to common forms of adult-onset glaucoma with complex inheritance also explains only a fraction of the disease heritability.² Unlike early-onset diseases where a single mutation can be responsible for the disease, in complex disorders each genetic variant has a small incremental effect on disease susceptibility. Aggregates of multiple gene variants, possibly in combination with environmental risk factors, are required for the disease to become fully manifest. Disease genes influencing susceptibility to adult-onset disorders are identified by genomewide association studies where the frequency of gene variants is compared in cases and controls. Because gene variants contributing to adult onset diseases with complex inheritance have relatively modest biological effects, the difference in variant frequency between cases and controls is usually small. Consequently, gene discovery for adult-onset disease-associated variants requires a large number of cases and controls providing sufficient statistical power to detect significant differences in variant allele frequencies. The current collection of genes known to contribute to adult-onset forms of glaucoma were discovered using thousands of cases and controls and finding new genes will require even larger sample sizes with more statistical power. Assembling the large cohorts required for these studies typically requires collaborative efforts and several consortia have formed to facilitate GWAS studies in glaucoma including the NEIGHBORHOOD (National Eye Institute Glaucoma Human genetics collaboration Heritable Overall Operational Database) and the IGGC (International Glaucoma Genetics Consortium) consortia. Additionally, all the genes and genomic regions discovered so far for adult-onset forms of glaucoma have been in the Caucasian and Asian populations. Genetic studies of African-Americans, the ethnic group most likely to develop glaucoma, are clearly needed.

The GWAS is an initial approach that identifies common variants that contribute to disorders with complex inheritance. Other analyses to consider for complex disorders such as POAG, NTG, pseudoexfoliation syndrome and angle closure glaucoma are gene-gene and gene-environment interactions and biological pathway analyses. Environmental exposures are emerging as important risk factors for pseudoexfoliation syndrome and glaucoma and considering the robust association of pseudoexfoliation syndrome with the LOXL1 gene, studies of gene-environment interactions involving LOXL1 will be important. Biological pathway analyses are also proving to be useful in identifying molecules and pathways that contribute to disease and that may be targets for novel therapies.³

Genetic testing

Currently, the eight genes known to cause early-onset familial forms of glaucoma can be used for genetic testing. Genetic testing for these disorders has several benefits: mutation carriers can be identified and treatment can be started before damage to the optic nerve occurs, in families disease surveillance can be targeted to mutation carriers so that family members who do not have the mutation and are not at risk can avoid unnecessary exams, and as gene-based therapies are discovered individuals who are carriers of a specific mutant gene who would benefit from gene-based therapy can be identified.

Genetic testing should be performed in a CLIA (Clinical Laboratories Improvement Amendments) certified laboratory where quality control can be monitored. Testing requires either a saliva or blood sample from the patient and family members. Most genetic testing uses DNA sequencing to detect disease-causing mutations and this may be done on a gene by gene basis or by screening all the genes that can contribute to the condition in one test using next-generation sequencing techniques.4 After the test data is analyzed a report is written and returned to the referring physician for discussion with the patient and families and genetic counseling as necessary. Patients and their families who are most likely to benefit from genetic testing using the current collection of genes are those with disease onset before age 40 or with advanced disease before age 50 and a history of glaucoma in a first degree relative.⁵

For adult-onset glaucoma gene-panel tests, allowing for simultaneous testing of multiple risk alleles, are a better measure of adult-onset disease risk. However, to be effective the gene panels need to be comprehensive and the clinical benefit of the test should be evaluated. The genes currently known to contribute to the adult-onset glaucomas represent only a fraction of the overall heritability and panel tests have not yet been studied to determine if testing for this collection of genes has clinical utility. New studies identifying more genes could lead to more comprehensive gene panels that have the potential to accurately identify patients at risk for disease prior to the onset of irreversible damage to the optic nerve.

Gene-based therapies

An important goal of genetic research is to define the underlying molecular events responsible for the disease and use that information to develop novel gene-based therapies that target the actual disease-causing molecular pathophysiology.

An important goal of genetic research is to define the underlying molecular events responsible for the disease and use that information to develop novel gene-based therapies that target the actual disease-causing molecular pathophysiology.

For some types of early-onset familial glaucoma gene-based therapies may be available in the near future. For example, patients with elevated IOP due to mutations in MYOC may benefit from chemical chaperones or other approaches that relieve the misfolded protein response induced by gene mutations.⁶ The discovery of more genes responsible for early-onset glaucoma will make it possible to offer gene-based therapies to more patients affected by these devastating forms of glaucoma.

For adult-onset glaucoma there is also the promise of gene-based therapies in the future. Rather than targeting a single gene, for these complex forms of glaucoma it may be preferable to target the biological pathways that include the disease susceptibility genes. The NEIGHOBRHOOD study has identified several pathways associated with POAG and NTG.3 Developing therapies directed at these pathways may lead to novel and potentially neuro-protective treatments. The discovery of more genes contributing to adult-onset forms of glaucoma will likely identify additional important disease mechanisms that could also be targeted for novel therapeutics.

Over the past decade there has been considerable progress in our understanding of the genes responsible for and contributing to both rare and common forms of glaucoma. An important next step is the discovery of new genes making it possible to gain a better definition of disease-related molecular pathophysiology, improve genetic testing and provide more opportunities for gene-based therapies.