Investigating the influence of genetic variation associated with age-related macular degeneration on plasma levels of complement regulatory proteins

Age-related Macular Degeneration (AMD) is the leading cause of blindness in the Western world. Recently, it has become clear that the genetic make-up of an individual has a strong influence on their risk of developing AMD. Gene variants have been found which are 'protective', i.e. individuals with these variants have lower risk of AMD, and which are 'causative', i.e. their presence results in increased risk. There are two main genetic regions that modify AMD risk. One, on chromosome 10, is poorly understood. The other, on chromosome 1, provides the code for a series of proteins called Complement Factor H (FH), Factor H-Like Protein 1 (FHL-1), and five proteins called Factor H-Related 1-5 (FHR1-5). These proteins are involved in regulating part of the immune system called the complement cascade. It is thought that genetic variations on chromosome 1 alter the amounts of these proteins, resulting in inflammation in the eye and ultimately to the development of AMD. This is supported by a number of studies where the absence of one of these proteins, or a change in its sequence (hence function) can be shown to change the risk of AMD occurring.
While it is assumed that these AMD-associated genetic variants change the levels of FH, FHL1 and FHR1-5 present in the body, this has been difficult to confirm. These proteins are very similar (indeed FHL-1 is identical to the first half of FH, with the exception of a very small tag at one end). This means that standard methods, which recognise and measure a protein based on its shape are fraught with problems, as it is very difficult to prove exactly which form is being measured. In this project, we will take a different approach - mass spectrometry (MS). MS works by 'weighing' the molecules of interest, then counting how many of each are present. Since we know the structure of these proteins, we can calculate their mass and detect them with very high confidence. Indeed, MS is now routinely used for testing blood levels of many compounds as part of routine clinical care. We have developed an MS-based method which can measure the levels of all seven proteins of interest in plasma at the same time. This method will be of great value. We will use it to analyse blood samples where we have already used standard methods and see how the results match, showing which methods are reliable. Secondly, we will measure the levels of these seven proteins in plasma collected as part of a large genetic study of AMD. The donors of these blood samples have already had their genetic profile determined. By measuring the amounts of protein, we can compare with the genetic data and see for the first time how the genetics impacts on the levels of each protein, and in turn how the levels of these proteins confer AMD risk. This research will provide new understanding as to how AMD develops, will improve our ability to define risk, and may allow the development or monitoring of new treatments.
This assay will also be useful for research into other diseases. These proteins are known to be important for certain kidney diseases and reliable ways to measure them will be important both for research and clinical care. There is also evidence that they contribute to other conditions such as Alzheimer's disease.

Our objective is to measure plasma levels of Complement Factor H, Factor H-like Protein 1, and Factor H-Related proteins 1-5, encoded by a region of chromosome 1 where gene variants modulate risk of developing Age-related Macular Degeneration (AMD). It is hypothesised that these variants modulate protein levels leading to dysregulation of the alternative complement pathway and thereby driving AMD pathology.
Immunoassays for these proteins are prone to interference from non-specific protein detection. We have developed a targeted liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM-MS) method to measure all seven gene product from this region. In this project we will characterise this assay using guidelines laid down by the European Medicines Agency to assess its accuracy for analysis of large numbers of clinical samples, and subsequently deploy it in two related studies. The first will compare data generated using the SRM-MS assay to that generated by existing ELISAs on the same samples to determine the specificity and quantitative accuracy of the immunoassays. The second will measure the levels of these seven proteins in a cohort of 662 individuals from the MRC-funded Cambridge AMD Study for whom full genotyping data is available. Protein level and genotype will be correlated. Since we will be measuring all seven proteins simultaneously, we will be able to study the effects of stoichiometric ratios of these regulators on pathogenesis for the first time.

Ultimately many groups of people may benefit from this research. This includes patients with AMD, where we expect our research will determine the role of the FH, FHL-1 and FHR proteins in disease development, therefore providing new data from which to derive improved risk-prediction models and, via both our targeted and untargeted approaches, identify new targets for therapy and/or potentially a means by which therapeutic intervention can be monitored.
However, other patient groups stand to benefit also. Factor H is reportedly involved in the development of several renal disorders, and has been proposed as a marker by which it may be possible to track the development and severity of Alzheimer's disease, for example. The work carried out in this project can be applied to other projects in these areas, pushing our knowledge of the disease process and providing tools for prognosis and disease management.
This research is challenging. There are few academic groups worldwide who have to date characterised mass-spectrometry based assays for use in determining proteins to the level we propose here. However, this work is critical for new biomarkers either emanating from large discovery studies or which are already known from hypothesis-driven research to begin to be translated into large numbers of clinical samples for assessment. This project will have positive impact on this field, equipping the staff working on this project with a highly sought after skills and experience base and demonstrating the ability of these methods to deliver plasma protein biomarker measurement in the absence of specific antibodies for the development of improved care across the spectrum of human disease. This will have positive impacts on the development of biomarker measurement science in both academia and industry, and will enhance the reputation of the UK in this field.