Dr Willoughby’s research theme is Molecular and Regenerative Medicine with a strong translational basis. His research focuses on ocular degenerative diseases many of which are associated with ageing. The main aim of this research is to improve the understanding, diagnosis and treatment of the major worldwide blinding disorders such as corneal blindness, glaucoma, retinal degenerations and vascular retinopathy. To do this a variety of clinical and experimental approaches are used including genetics, molecular biology and cell biology.
Currently, Dr Willoughby’s research is concentrated in two main areas:
1. Degenerative disorders of the cornea.
The cornea is the major refracting structure of the eye and its clarity, shape and function is central to vision. Disturbances of the cornea, which include keratoconus, endothelial dysfunction, stem cell failure and corneal scarring, are a common cause of world blindness in both developed and developing nations. Many of these conditions have a strong genetic basis and require corneal surgery. This facilitates molecular biology, gene mapping and cell function studies into their pathogenesis and treatment. Our group is performing genetic analysis and gene expression studies in keratoconus and endothelial dystrophies in collaboration with other groups in the UK, Europe, Australia and India and utilises a variety of research techniques.
Figure 1. Corneal topography in keratoconus showing ectasia and protrusion of the cornea in 3D
Figure 2. Cultured keratocytes showing vimentin positive staining
The ectodermal dysplasias are a heterogeneous group of disorders affecting the development of the ectodermal structures in the body including the eye. Visual impairment is a major cause of morbidity in keratitis-ichthyosis and deafness (KID) syndrome, ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome and ankyloblepharon-ectodermal dysplasia- clefting (AEC) syndrome. Both EEC and AEC result from p63 mutations. Our group was previously involved in the genetic investigation of KID syndrome and p63 disorders, and have been studying the ocular phenotype using impression cytology, stem cell analysis and microbial multiplex PCR. The ocular phenotype in EEC and AEC results from defective p63 which is a key protein in regulating the corneal stem cell population. In collaboration with the Veneto Eye Bank Foundation, Venice, Italy, we are developing the application of genetically modified corneal stem cells to correct limbal stem cell failure in EEC syndrome. This approach could then be applied to other genetically determined corneal epithelial disorders like aniridia and potentially keratoconus. Understanding the role of corneal stem cells in health and disease is central to understanding and developing therapeutic strategies for corneal blindness.
2. Degenerative disorders of the retina.
Retinitis pigmentosa and retinal dystrophies are a genetically, heterogeneous group of conditions. One of the major hurdles in clinical ophthalmology and genetics is obtaining a genetic diagnosis. This has clear translational benefits and is of great concern to many of the patients seen in the Ocular Genetics Service. Specifically, a genetic diagnosis facilitates counselling, gives information on genetic risk and provides a more precise clinical prognosis. It is also a crucial pre-requisite to human gene-specific clinical trials and can inform the patient of current treatment options. In order to advance genetic testing (in collaboration with Dr David Simpson and Miss Giuliana Silvestri) we have developed a next generation sequencing approach in inherited retinal degeneration. Using this technique we sequenced all exons on the X-chromosome and within the whole genome to identify novel genes associated with retinal degeneration. This powerful approach can identify new therapeutic targets and pathways involved in genetic retinal disease. We are applying our experience with next generation sequencing to gene identification in keratoconus and gene expression analysis in the keratoconic cornea and retina in model systems of glaucoma and ischaemic retinopathy.
|Figure 3. Illustration of massively parallel sequencing analysis of RPGR performed using the Illumina Genome Analyzer (GA) II in a DNA sample from a patient with a known RPGR sequence variant (RPGR exon 14 c.1598G>A p.T533M). The target regions tiled across the RPGR gene exons in our Nimblegen sequence capture array are shown as a custom user track in the UCSC Genome Browser. Below this is the coverage for each exon in numbers of NGS reads from one sample. The reference sequence and consensus from alignment of the NGS reads are shown for exon 14. The coverage varied from 500-709 in this region and some of the reads which span a known variant are visible. This G/A heterozygote was confirmed by conventional Sanger sequencing (bottom). Courtesy of Dr David Simpson.|
Glaucoma is the second commonest cause of blindness worldwide and accounts for about 10% of all blind registration in the UK. With an ageing population, the prevalence of glaucoma is expected to increase by 30% in the next 20 years. Primary open-angle glaucoma (POAG) is the most common form of glaucoma and has a strong genetic basis. Our group is using a number of strategies to investigate the genetic basis of glaucoma from study populations in Ireland, the UK and India. These include genome wide SNP microarrays and DNA pooling (SNP-MaP), proteomics directed candidate gene sequencing and next generation sequencing. Using model systems we are also analysing gene expression and alternative splicing in the glaucomatous and ischaemic retina. Analysis of EPO signalling in these systems has highlighted splice variants as potential therapeutic targets for neuro- protection and angiogenesis.