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

Lead Research Organisation: University of Manchester
Department Name: School of Medical Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Clark SJ (2018) The eye as a complement dysregulation hotspot. in Seminars in immunopathology

 
Title FH, FHL-1 and FHR1-5 assay 
Description This is a mass spectrometry based assay for the detection of circulating complement factor H, Factor H-like 1 and the five Factor H Related proteins in plasma. This assay has been validated and is being deployed at the time of writing across a cohort of 700 patient samples. A Patent application to protect novel aspects of this method is also in progress. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact The assays is currently being deployed within a 700-patient cohort to assess the effect of AMD-associated SNPs on the expression/circulating levels of these proteins. We are currently blinded to the results and will process in due course. We have also successfully used the assay as part of a collaboration looking at FHL-1 levels in the plasma of patients with glioblastoma. 
 
Description Dr Simon Clark/Prof Paul Bishop - Complement in the Eye 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Dr Clark, prof Bishop and I are collaborating on several project regarding complement regulation at the back of the eye. To this collaboration I bring expertise in protein biochemistry, mass spectrometry and protein quantitation. Specifically this collaboration is focussed around 2 projects: Project 1) Measurement of complement proteins, including FH, FHL-1 and FHR1-5 in tissues and biofluids, and Project 2) The development of novel complement mediators for potential therapeutic use. My specific contributions to these are: Project 1) Design of a protocol which is capable of discriminating between, and subsequently quantifying homologous complement factor H family members using mass spectrometry. We now have MRC funding to build this assay. Project 2) Aid in the design of two novel polypeptides (patent filed Jun 17 and Jan 18 respectively), including selection and characterisation of specific features, characterisation of said polypeptides by mass spectrometry and, going forward, the development of methods to quantify these in biological matrices. We are currently working together to develop a pathway to Phase I clinical trials for both compounds.
Collaborator Contribution Dr Clark brings significant expertise in complement biology, and growing reputation in the complement field. His contribution to our projects includes Project 1) Expertise in complement biology, provision of standards and materials, access to other sample cohorts via his existing collaborations. Project 2) Simon is leading this project and provides biological expertise and mechanistic insight, design of the novel polypeptides and their production and biological characterisation. Simon is also lead on discussions with potential collaborators or investors into this project. Prof Bishop is a clinical ophthalmologist and an expert in eye disease, in particular Age-Related macular Degeneration. His contributions to these projects include: Project 1) Access to samples and expert knowledge of AMD genetics which will be critical in respect of data analysis. Project 2) Paul has experience in patient selection and clinical trial design in AMD. he also has experience from previous projects of the development of novel therapeutics in this patient groups and provides critical strategic input into the direction of this project.
Impact This is a multidisciplinary project between myself (protein biochemistry, mass spectrometry, analytical chemistry), Dr Clark (complement biology, Age-related macular degeneration) and Prof Bishop (Clinician, AMD genetics and pathophysiology, clinical trials). Outputs thus far include an MRM Confidence in Concept award and two patents, filed June 17 and Jan 18.
Start Year 2014
 
Title C3B BINDING POLYPEPTIDE 
Description Polypeptides comprising a C3b binding region are disclosed, as well as nucleic acids and vectors encoding such polypeptides, and cells and compositions comprising such polypeptides. Also disclosed are uses and methods using the polypeptides for treating and preventing diseases and conditions. 
IP Reference WO2019138137 
Protection Patent application published
Year Protection Granted 2019
Licensed No
Impact We are in the proces of spinning out a compant based on this ip, with the aim to incorporate in mid 2020.
 
Title C3B INACTIVATING POLYPEPTIDE 
Description Polypeptides comprising a C3b bindingregion and a C3d inactivating region are disclosed, as well as nucleic acids and vectors encoding such polypeptides. Also disclosed are cells and compositions comprising such polypeptides, and uses and methods using the same. 
IP Reference WO2018224663 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact This discovery is currently the subject of a commercial discussions with several interested parties
 
Description I'm a Scientist - Careers Zone 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Following a successful event during the formal I'm a Scientist... fortnight, I have since stayed in contact with this projects as part of their Careers Zone. This involves taking part in careers-focussed web-chats with schools, and answering questions submitted online by pupils wanting to know more about careers in science.
Year(s) Of Engagement Activity 2015,2016,2017,2018,2019
URL https://imascientist.org.uk/
 
Description I'm a Scientist get me out of here 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I'm a Scientists is a national event where scientists compete in 'Zones' , taking part in web-chats with schools and answering submitted questions over a 2 week period. During the second week, schools vote out the scientists in a series of eliminations until one remains. I took part in the Ageing Zone, discussing a range of topics including what happens when we age, alongside age-related diseases such as dementias, finishing runner-up in my Zone and answering more questions than any other scientists (over 300!) across the fortnight.
Year(s) Of Engagement Activity 2015
URL https://imascientist.org.uk/
 
Description Science Spectacular 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Science Spectacular is an annual event held at the University as a Science Fair where many groups present stands with activities to explain and promote their research. My lab stand is based around blood tests and detecting biomarkers, to use either as diagnostics or to identify new biological disease mechanisms and therapeutic targets, allowing us to communicate across the range of projects we do. Due to having several non-native English speakers as volunteers, we have also delivered the activity on both Arabic and Polish to visitors, as well as in English.
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016,2017,2018