Triplet repeat expansion-associated inherited corneal disease: from genetic mechanism to clinical consequences

Fuchs endothelial corneal dystrophy (FECD) is a common vision-impairing disease that affects 4.5% of the population over 50 years of age. Both eyes are generally affected, and this is because the cornea, the transparent window at the front of the eye, loses its ability to remove excess fluid from its outer layers and becomes swollen and cloudy. The only effective treatment for advanced FECD is corneal transplantation. In the UK, 80% of the FECD cases are caused by a common genetic disorder called a 'CTG18.1 repeat expansion', where this short sequence of genetic code repeats and expands to a threshold that causes disease.

FECD is the most common reason for corneal transplant in the UK. However, the surgery is costly, and there is a global shortage of donor tissue. In an effort to reduce the demand for corneal tissue, multiple innovative treatment options are emerging, including a gene therapy developed at our lab. The long-term success of these therapies will rely on identifying individuals at risk of developing FECD and initiating early treatment before irreversible damage occurs. Having a better understanding of the genetic mechanism in FECD would help to achieve these.

In other better-understood diseases caused by abnormally repeated genetic codes (eg. myotonic dystrophy), the more the disease-causing genetic code is repeated, the earlier the onset of disease and the more severe symptoms patients will have. Moreover, the number of these repeated genetic codes is larger in the affected tissues. We also know that in these conditions, the number of the disease-causing genetic code increases when the disease is passed down from one generation to the next, resulting in earlier and more debilitating disease in each succeeding generation. This project aims to find out whether these clinically important patterns also occur in FECD, which is currently a poorly understood disease.

Our study will take place in the laboratory setting at UCL Institute of Ophthalmology, where we have already performed extensive genetic analysis of a large group of FECD patients.

Aim 1. I will generate new genetic data and use statistical models to examine the relationship between the number of CTG18.1 repeats of our recruited patients and their age when they require a corneal transplant. Following my preliminary work, I expect to see a trend where patients with larger CTG18.1 repeats require corneal transplants earlier. This knowledge will enable us to predict how fast disease will progress based on genetic information.

Aim 2. I will use an innovative technique called Single Molecule (SM)-PCR to analyse the DNA in the affected corneal tissues removed from FECD patients during corneal transplant surgery. Due to technical limitations, analysis of diseased corneal cells has not been successfully performed before. As the cornea is the only tissue affected by FECD, I predict the number of CTG18.1 repeats in corneal cells may be a lot larger than in the blood cells of the same patient.

Aim 3. I will analyse the DNA of FECD patients' siblings to assess the proportion of individuals carrying the CTG18.1 expansion that also develop FECD. Adult children of FECD patients will also be recruited to discover how the repeats behave (i.e. if it gets bigger or smaller) when passed onto successive generations. This will enable us to have a better understanding of how CTG18.1 changes within families and its risk associated with FECD.

Currently, it is not known how the disease-causing gene of FECD modifies its clinical features. This important research will help us gain more in-depth knowledge about FECD, and potentially allow doctors to give more accurate genetic counselling on the progression of FECD and its risk implication to the next generation of FECD patients. Knowledge of CTG18.1 repeats in affected corneal cells may also open up research for new treatments options.

Fuchs endothelial corneal dystrophy (FECD) is the most prevalent repeat expansion disease in humans. There is increasing evidence that somatic expansion controls clinical severity of repeat mediated diseases, though this is yet to be explicitly elucidated in FECD.

Aims:
1) Quantify the role of CTG18.1 allele length in driving progression of FECD: In addition to recruited probands already being genotyped by conventional PCR-based technique and the next-generation MiSeq, I will apply the innovative single molecule PCR (SM-PCR) techniques to generate the CTG18.1 allele length in probands whose allele distribution fall beyond the detection threshold of existing methodologies. Leveraging extensive data from this unique patient cohort, I will use regression models to examine the correlation between allele lengths and the age of corneal transplantation, a surrogate marker for disease severity.

2) Determine the tissues-specific CTG18.1 expansion in affected corneal endothelial cells: For the first time, SM-PCR will be applied to probe the CTG18.1 allele length distribution within affected endothelium removed during surgery and compare to patient-matched blood-derived DNA to determine heterogeneity in different tissues. FECD corneal endothelial cells are expected to harbour larger CTG18.1 expansion than blood derived samples.

3) Characterising intergenerational instability of CTG18.1 and variable penetrance of FECD: Siblings and children of our FECD probands will be recruited for genotyping. Genotyping data of the siblings will be harnessed to characterise the variable penetrance of FECD. Intergenerational variation of CTG18.1 derived from comparing allele lengths of parents and offspring will provide novel insight into the (in)stability of CTG18.1 expansion through the germline.

Outcomes: Enhancing our understanding of the genetic modifiers on the clinical consequences of FECD may augment risk stratification approaches and allow for personalised medicine.