The role of DNA methylation in stratifying and understanding paediatric nephrotic syndrome - a combined bioinformatics and CRISPR-cas9 approach

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Nephrotic syndrome: Nephrotic syndrome (NS) is a condition characterised by the kidneys leaking huge amounts of protein into the urine, significant fluid retention and low protein levels in the blood. Children who suffer from NS can develop kidney failure and require lifelong treatment; therefore, this condition has a drastic impact on their lives. Our knowledge of NS is increasing and we now recognise that it is not one single disease, which explains why people with NS can exhibit markedly different responses to the same treatment and why some people's NS will return after they have had a kidney transplant, but for others it will not. Findings from research studies suggest that NS consists of 4 conditions (subgroups) and that the disease may be driven by a different mechanism in each subgroup. Recent advances in genetics have identified that abnormal DNA sequences in genes which are important for kidney function are the cause of NS in one of the subgroups. However, the causes of NS in the 3 remaining subgroups are unknown and there is no clinically useful method to identify which subgroup a patient belongs to.DNA methylation: DNA methylation (DNAm) is one of the body's molecular mechanisms for switching genes on and off. A person's DNAm profile can change throughout their lifetime in response to their behaviour and their environment. Therefore, DNAm may play an important role in disease development. DNAm profiles have shown great promise in distinguishing between different disease subgroups, for example, DNAm profiles can be used to accurately classify patients with different types of brain tumours. I studied blood DNAm profiles from 105 people with NS to explore whether this mechanism may be important in NS. I was fascinated to find that people in the 4 NS subgroups had different DNAm patterns; also, the differences pointed to specific genes which could be important in NS but have not previously been examined. I will build on these findings by studying DNAm profiles and genetic data from 280 NS patients and by using cutting-edge computer science and laboratory techniques. The aim and importance of my research: My work aims to classify NS patients into the 4 NS subgroups by using their blood DNAm profiles. A blood test that can identify which subgroup a patient belongs to is the first step towards personalised NS treatment; it would allow children with NS to avoid futile and potentially dangerous medication regimes and focus on beneficial treatments. It could also be used to predict which patients may develop NS after kidney transplantation and which would not. By studying the molecular profiles of people with NS, I will also gain a new understanding of the separate disease pathways of each subgroup, which could lead to novel targeted NS treatments. There are 3 stages to my research:1. Use blood DNAm profiles to segregate the 4 NS subgroups.I will use sophisticated machine learning techniques to determine whether blood DNAm patterns can consistently and accurately segregate patients into the 4 subgroups. 2. Examine whether differences in DNAm are likely to cause NS.I will compare blood DNAm profiles between people in the 4 NS subgroups. By analysing DNAm profiles alongside genetic data I will tease out which DNAm differences are more likely to be causing NS; this is crucial for prioritising which specific sites of differing DNAm have the most potential to become treatment targets.3. Alter DNAm in a targeted manner in blood cell lines.I will alter DNAm in blood cells at the 5 most promising DNAm sites identified in stage 2 and study the effect that this has on gene regulation and protein production. To do this, I will learn highly specialised laboratory techniques which utilise gene editing technology. Ultimately, this work is the first step towards creating new NS treatments which target DNAm.