Investigating the molecular mechanisms influencing therapeutic responses in glaucoma

Supervisors: Professor Jane Sowden and Dr Daniel Kelberman

Glaucoma in infancy and childhood is a potentially blinding condition affecting 1 in 18,500 children in the UK and accounting for 5% of blindness in children worldwide. The condition usually presents as raised intraocular pressure (IOP), which if left untreated, causes optic nerve damageleading to blindness. IOP is regulated by a balance between the production and drainage of aqueous humor inthe eye. The majority of aqueous drainage occurs through a sieve like structure called the trabecular meshwork (TM) located in the angle region between the cornea and iris. To date, no conclusive mechanism has been identified as being responsible for elevated IOP in glaucoma patients, but abnormal drainage of aqueous has been implicated.The only proven effective treatment for glaucoma to date is the reduction of IOP. Within the last 15 years prostaglandin (PG) analogues, have become most commonly used as first line therapy (Lee and Goldberg. 2011). However, knowledge of the molecular mechanism of action these drugs is still elusive, and some patients fail to respond to medical therapy requiring surgical intervention. 50% of cases requiring surgery exhibit advanced visual field damage andit is not understood why some patients fail to respond adequately. We hypothesise that functional variation in genes involved in the uptake or response to PG analogue treatment in cells involved in IOP regulation may influence the effective response of an individual to pressure lowering medication.

Recent innovations in next-generation sequencing technology (NGS) have offered an as yet unparalleled advancement in the analysis of gene expression by permitting high-throughput sequencing of entire cDNA libraries (RNA-seq) (Mortazavi et al., 2008; Sultan et al., 2008) permitting the detection of rare transcripts, as well as the analysis of novel splice variants.This project will use NGS technologies to characterise the molecular response of prostaglandin analogues in cells relevant to IOP regulation. Primary cell cultures will be treated with a range of PG analogues currently used in the treatment of elevated IOP.By using RNA-seq the student will identify genes that alter their expression or alternative splicing patterns in response to drug treatment. Bioinformatic analysis of these data will identify candidate genes involved in drug response pathwaysintissues within the eye responsible for maintaining normal pressure. Candidate genes will be screened for genetic variation in patients that failed to respond adequately to pressure lowering drugs as compared to those that do. Candidate variants will be analysed using a range of bioinformatic approaches to assess their effect on protein function. Those identified exclusively in the non-responder cohort will be prioritised for follow-up using in vitro molecular biology assays to identify their role in drug response pathways. The project will involve using a variety of molecular genetic and molecular biology techniques including RNA-sep, mutation analysis using NGS and/or Sanger sequencing, bioinformatic prediction of the functional effect of genetic variation and cell culture techniques.

This analysis will provide for the first time a comprehensive and detailed insight into the molecular responses of both coding and non-coding RNAs and their interacting pathways active in tissue within the eye relevant to IOP physiology in response to currently used topical glaucoma medications. This will provide a better understanding of the actions of these treatments to modulate outflow and the potential to begin to unravel why some patients do not respond effectively to PG analogue therapy. The identification of subĀ­groups of patients unlikely to respond to certain therapies will guide personalised patient management to reduce prescription of ineffective treatments and guide effective and timely clinical care to prevent visual loss.

References:
1) Lee AJ, Goldberg I. Expert Opin Emerg Drugs 2011;16:137-61.
2) Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Nat Methods 2008;5:621 -8.
3) Papadopoulos M, Cable N, Rahi J, Khaw PT. Invest Ophthalmol Vis Sci 2007;48:4100-6.
4) Sultan M, Schulz MH, Richard H, Magen A, Klingenhoff A, Scherf M, et al. Science
2008;321:956-60.